Volume 4 | Issue 1 Article 2

1941 Inclusion Bodies Alfred M. Lucas Iowa State College

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Recommended Citation Lucas, Alfred M. (1941) "Inclusion Bodies," Iowa State University Veterinarian: Vol. 4 : Iss. 1 , Article 2. Available at: https://lib.dr.iastate.edu/iowastate_veterinarian/vol4/iss1/2

This Article is brought to you for free and open access by the Journals at Iowa State University Digital Repository. It has been accepted for inclusion in Iowa State University Veterinarian by an authorized editor of Iowa State University Digital Repository. For more information, please contact [email protected]. Inclusion Bodies How specific are inclusion bodies for the identification of virus diseases? Alfred M. Lucas, A. B., Ph. D.*

N ORDER that we may both have in long to the pox group and a steady accu­ I mind the same thing during this paper mulation of facts for the past 35 years has it is probably well to set forth a brief now culminated in a reasonably satis­ description of inclusion bodies. In a broad factory understanding of the relationship biological sense the term applies to any of virus to inclusion body for this group formed mass of material, such as secretion of diseases. The development of this granules and plastids, but it is not in this sense that the pathologist uses the term; TABLE I for him it has come to mean those masses of material which are associated with virus Viruses Producing Inclusions In: diseases. Those in the cytosome, when Cytosome Only they are small, have practically no unique Vaccinia Sheep pox characteristics which will readily dis­ Fowl pox tinguish them from cytoplasmic accumula­ Molluscum contagiosum tions of non-virus origin but as they grow Infectious myxomatosis of rabbits Trachoma larger their size becomes a valuable cri­ Rabies terion. Those in the nucleus are more Louping ill easily identified in that they are usually Nucleus Only acidophilic and they usually seek a posi­ Varicella (chicken pox) Vesicular stomatitis tion in the center of the nucleus. There Infectious warts of dogs they lie free from the chromatin which Yellow fever Rift Valley fever migrates toward the nuclear margin. The Virus III of rabbits existence of an object which appears to Disease of parrots and parrakeets be an inclusion body is not proof of the Herpes simplex B-virus presence of a virus but merely an indica­ Pseudorabies tion that a virus should be considered if Borna's disease of horses no bacterial agent can be found. If all Poliomyelitis Fox encephditis virus diseases produced a similar type of Jaundice of silkworms inclusion body the problem would be sim­ Cytosome and Nucleus ple in that one could seek out that element Variola (small pox) common to all of them; but some are Alastrim (mild small pox) Submaxillary gland disease of guinea associated with inclusion bodies in the pigs cytosome, some in the nucleus, some in of ground moles both nucleus and cytosome and many pro­ Equine encephalomyelitis duce no inclusion bodies at all. Repre­ No Cell Inclusions St. Louis encephalitis sentative examples are given in Table 1. Mumps Most of the virus diseases which pro­ Rous sarcoma of fowls duce inclusion bodies in the cytosome be- Sac-brood disease of bees Many plant viruses "Department of Zoology, Iowa State College.

Fall, 1941 11 knowledge will be reviewed briefly be­ reaction. It was this protecting substance cause it serves as a challenge to what we which made possible the separation of the should be able to do with the intranuclear inclusion from the cell by digestive en­ inclusions. Many years ago the elemen­ zymes and its absence in molluscum con­ tary bodies were discovered in smears and tagiosum (3) explains why the method it was suggested and later proved that would not work for this virus. It has these represented the actual virus agent. already been mentioned that corneal cells Their size was at the limit of microscopic add to the developing vaccinia inclusion visibility and filtration experiments have body a basophilic material (1) so that since showed that the poxes average interestingly enough we are led again to about 0.150 micron. When stained they the earliest interpretation of cytoplasmic are considerably larger than this. With inclusions expressed by Prowazek when the ultra-microscope objects as small as he named them Clamydozoa and explain­ 0.1 micron may be distinguished and ed that it was a cloaked organism-that the new electron microscope will probably the cell contributed a mantle over the make some of the smaller viruses visible. infective agent. Thus, for the pox group But to identify particles as small as these of viruses one can say that finding an within a cell and be convincing about it, inclusion body is comparable in diagnos­ is rather more difficult. Had the granules tic importance to finding the organism in remained uniformly dispersed in the cell anthrax or tuberculosis. they probably would not have been rec­ Not all cytoplasmic inclusions have re­ ognized. One of the early cytological stu­ vealed their secrets so well and the oppo­ dies (1) of vaccinia virus came to the site extreme is represented by the Negri conclusion that the basophilic inclusion bodies associated with rabies. The lyssa body was an aggregation of materials pro­ bodies and Negri bodies are sufficiently duced by the cell but that under the distinctive in their structural organiza­ stimulation of the virus abnormal amounts tion that they have been used satisfactorily were produced and these clumped togeth­ for diagnostic purposes but what they er. A contrary opinion (2) to this was are is still completely unknown as evi­ rendered for fowl pox when the cyto­ denced by the wealth of suggestions. Here plasmic inclusion bodies were separated are some: that the basophilic center is from the cell by digestive enzymes. The the organism and that the acidophilic inclusion bodies were washed repeatedly mantle is contributed by the cell; that the and then inoculated into the feather folli­ Nissl substance becomes modified into the cles of the fowl. One inclusion body Negri bodies; that the neurofibrils are would produce the disease. Efforts to converted into these bodies and finally handle the inclusion bodies of molluscum that they represent extruded nucleoli. contagiosum (3) in a similar manner were Their value for diagnostic purposes would not successful because the bodies fell probably be increased if one knew what apart into their separate elementary units. they are. Recently, by the growing of rabbit cornea Turning to the problem of the intranu­ in tissue culture (4), it has been possible clear inclusions we find that progress has to observe the multiplication of vaccinia been slow because the inclusions stain elementary bodies and their coalescence like oxychromatin which is a normal con­ into clumps within the cell. Consequently, stituent of the nucleus and because the there no longer is any doubt for this group idea that an organism would elect to re­ of viruses, at least, that the inclusion body side in the nucleus rather than in the is an aggregation of virus particles, but this cytosome has relatively little analagous is not the complete story; the cell contri­ data to support it. We do have the rickett­ butes something also. In fowl pox (5) it sias, however, as one good example of a was noted about 15 years ago that fats elab­ group of small organisms which are known orated in association with the Golgi ap­ to aggregate in the nucleus. Yet, in spite paratus moved toward the developing in­ of our limited knowledge on the subject, clusion body and gave to it a strong fat the idea developed that the presence of

12 The Veterinary Student an intranuclear inclusion was a fair indi­ of the published accounts are too sketchy cation that a virus was the etiological and very few photographs are sufficiently agent, even though the inclusion body in clear to show such small objects as part the nucleus may be merely a cell product. of a nucleus for the reader to form an Strong support for this idea came from independent opinion. Those papers, in the fact that none of the bacteria pro­ which the author has prepared drawings duced cellular reactions of this type and or even diagrams, are usually satisfactory. injections of various non-living substances Even examination of but one slide may usually failed to produce them. Then be misleading and it is often necessary to followed a period when many intranuclear make a fairly thorough study of the in­ inclusions were discovered apparently un­ clusion bodies with good optical equip­ associated with any virus disease. The ment to determine even the simple ques­ threatened agnosticism was circumvented tion of whether they are granular or amor­ when Cowdry proposed that intranuclear phous in type. inclusions be classified into two groups Of the two types, A has proven to be called type A and B (6). Type A included much more indicative of a virus than B those (a) which were granular and (b) (8) and the question always follows, "Are which produced a severe reaction in the the intranuclear inclusions aggregations cell leading to death. Type B were those of virus particles as they are in the poxes?" (a) which were amorphous irregular mas­ Attempts have been made to handle them ses or hyaline spheres and (b) which as Goodpasture did fowl pox but no suc­ produced only mild reactions in the cell. cess has as yet been reported (9). There­ It was unfortunate that two criteria were fore, our approach for the present must set up for each of the types because sev­ be indirect and the evidence circum­ erity of reaction and a peculiar morpholo­ stantial. gical configuration for the inclusion body The Beams ultracentrifuge has been a do not always go hand in hand. It has useful tool in this work because with it been suggested, therefore, that it would the substances within the nucleus along probably be more helpful to the patholo­ with the inclusion body may be stratified gist if types A and B connote merely their and thus it can be shown whether the respective morphologies and not indicate inclusion body has the same specific grav­ any degree of pathological reaction (7). ity as some constituent of the normal nu­ Under this plan types A and B would cleus or if it forms a distinct stratum of each include the following: its own. The granular type A inclusion produced by herpes simplex, which has Type A been inoculated into the corneal cells of Herpes simplex the rabbit, has been centrifuged and prov­ B-virus en to be lighter than any of the parts of a Yellow fever normal nucleus (10). The sequence of Rift valley fever layers from the lighter to the heavier pole Disease of parrots and parrakeets is, inclusion body, nuclear sap, and mixed Pseudo-rabies oxy- and basichromatin. The tentative Type B conclusion now reached and supported by Borna disease the work of Nicolau and Kopciowska (11) Poliomyelitis is, that in the case of herpes the particles Submaxillary gland disease of guinea pig, represent the virus elementary bodies. ground mole, and man Contra-wise centrifugation of several type Fox encephalitis B inclusions have always stratified them Canine distemper at the same level as the oxychromatin. Inclusions associated with some chemical This material separates out to form a layer agents between nuclear sap and basichromatin. This is true of fox encephalitis (7), sub­ This list is not complete for all of the maxillary gland disease of guinea pigs (12) described intranuclear inclusions. Many and inclusions produced by the subcutane-

Fall, 1941 13 ous injection of aluminum oxide (13). We which will aid the pathologist to analyze have some information on the infective better the inclusion-bearing cells which potency of these inclusions in the sub­ he sees in his routine or research prepara­ maxillary gland disease of guinea pigs. tions. For example, there are some data The potency is very low when the mono­ on the relative merits of tinctorial reac­ cytes bearing intranuclear inclusions are tions. We have no specific stains for in­ passed to uninfected animals (14) and tranuclear inclusions. The many tech­ becomes very high for the duct cells after niques in the literature, in most cases, the cytoplasmic inclusions make their ap­ merely make more evident things which pearance, but not while they contain only can be distinguished by most of the usual intranuclear ones. Further and still more staining procedures. For a while detailed convincing evidence that at least some of studies were made of the comparative the type B inclusions are not aggregations tinctorial reactions with a variety of of virus, that their presence does not auto­ stains but it has been shown in the sub­ matically indicate the existence of a virus, maxillary gland disease of guinea pigs and and finally that they may be merely ab­ especially well in fox encephalitis that the normal accumulations of oxychromatin, inclusion bodies may vary from eosino­ is furnished by the experiments of Olitsky philic to basophilic, that the inclusion is and Harford (15,16). They produced composed of two parts and that the reac­ characteristic type B inclusions by sub­ tion merely depends upon the proportion cutaneous injections of aluminum oxide of the two staining elements. On the other and hydroxide, iron hydroxide, carbon hand, type A inclusions are more consist­ and various other substances. They elim­ ently eosinophilic. The analysis of inclu­ inated the possibility of a latent virus. sions by microchemical techniques, thy­ Centrifugation of inclusions produced by monucleic acid reaction, test for masked aluminum oxide (13) gave the same strat­ iron, Millon test, microincineration and re­ ification series as for type B inclusions actions to various chemicals have been re­ associated with virus agents. viewed in publications by Cowdry and by The production of inclusions by chemi­ Findlay. cal agents is not new. It has been associ­ More important than staining reactions, ated with several heavy metals (17,18,19), per se, is the use of stains to differentiate arsenic, mercury, and lead, and the in­ and to follow the reactions of oxy- and clusions produced all belong to type B. basichromatin within the nucleus. It is In only one case has it been claimed that this aspect of cytopathology which seems a type A inclusion was developed by a to have been neglected, except in a few non-virus agent. Belt (20) found in the cases, and which should in the end be of liver of persons who died from severe some practical value to the clinician. burns that the intranuclear inclusions and The most important point to be empha­ Councilman-like bodies in the cytosome sized in our thinking about the cytopath­ were indistinguishable from those pro­ ology of inclusion body formation is to duced by yellow fever. He suggested the separate cellular reactions in conjunction toxin as a possible cause for the produc­ with the inclusion body formation from tion of the inclusion body but a number reactions leading to the death of the cell. of years ago Findlay noted that cells in The formation of an inclusion body does yellow fever looked as if they had been not automatically lead to cell death but dehydrated and the work of Underhill and the confusion occurs from the fact that the his associates (21) have shown that sev­ virus agent which may stimulate the de­ ere burns may cause the formed elements velopment of the inclusion body, often at of the blood to be concentrated as much the same time, produces an area of tissue as 38 percent and become so viscous that destruction so that the inclusion-bearing the blood fails to flow through the vessels. cells may be involved. A1thou~h the information of the true Many descriptions of inclusion body nature of the inclusion bodies progresses formation merely mention the presence of slowly yet contributions can be made (Continued on page 45)

14 The Veterinary Student Lt. W. S. Monlux, '37, formerly in the brane. After the chromatin reaches the Pathology Department at Cornell Univer­ margin, it reverses its movements and sity, is now stationed at East St. Louis crosses the halo to rejoin the inclusion (Collinsville), Ill. body to which it attaches so firmly that Dr. Robert L. Alkire, '36, formerly with centrifugation will not separate it. It re­ the B. A. 1. at Cumberland, Md., is now quires ten days to two weeks for the duct at Station Hospital, Langley Field, Va. cells to attain this condition. They be­ Capt. F. M. Bolin, '29, is serving with come greatly hypertrophied in the process the Vet. Provo Med. Sq., 2nd Cav. Div., and it is significant that the cell maintains Fort Riley, Kan. Dr. Bolin was formerly a normal nucleo-cytoplasmic ratio (if one with the Veterinary Dept., North Dakota deducts the volume of the inclusion body Agricultural College, Fargo, N. D. from the nucleus). In fox encephalitis a Dr. L. E. Stanton, '25, formerly at Jack­ peculiar reaction occurs in the early son, Minn., is now located at the Medical stages. At the time when the basichroma­ Field Service School, Carlisle, Pa. tin is marginating toward the nuclear The address of Lt. R. W. Rushmore, '39, membrane, the oxychromatin separates formerly of Columbus, Iowa, is now 824 from it and moves in the opposite direc­ Richmond Ave., Silver Springs, Mr. tion, namely, toward the inclusion body Pvt. Robert E. Storm, '40, is now serv­ which it encompasses. Thus, the inclusion ing with the Army at Camp Grant, Ill. body of fox encephalitis is not quite the same as that of the submaxillary gland Deaths disease although they are both of type B. Dr. Robert Miller, '38, was called to As a third example, the inclusions asso­ active duty in the army at Ft. Snelling, ciated with the subcutaneous injections of St. Paul, Minn., in July. Shortly after aluminum oxide resemble in their early being called to duty, a son was born to stages a plasmosome nucleolus but later Dr. and Mrs. Miller. Three days after become free of all basichromatin and are the birth of the son, Mrs. Miller died. Dr. Miller was to have been transferred to Carlisle Barracks, Carlisle, Pa. Dr. W. Wallace Boggie, '21, died Sept. Oil-0 -Sedative 22 at Madison, Wis. Dr. H. H. J onkers, '13, Three Rivers, A Soothing Intestinal Antiseptic Mich., died July 23, 1941. A Respiratory and Cardiac Stimulant Dr. G. F. Starkey, '91, died July 3, 1941. CONTAINS

INCLUSIONS (Continued from page 14) Beechwood Creosote • fIn a SUfficient amount an inclusion body surrounded by a halo Guaiacol of aromatic oil to and the chromatin as marginated against ~ucalyptus make further dilution the nuclear membrane. True, this is a unnecessary. conspicuous stage and one commonly Camphor j found but if the cell pathology is to be of further diagnostic value it is desirable to You'll Need This Preparation Many a Time Before Spring Comes Again have all of the events from the earliest to the latest, and whenever possible to have BETTER KEEP A GOOD SUPPLY ON HAND them separated from death changes. The Pint 55e Quart 85e Gallon $2.25 following remarks will indicate what is meant. When the inclusion body first ap­ Sample on Request pears in the submaxillary gland disease it hardly disturbs the normal organization J. ~. DeVine ldbordtories. Inc. of the nucleus. Then follows a breakdown Goshen of the linin net-work and the pulling away New York of the chromatin toward the nuclear mem-

Fall, 1941 45 cytologically similar to type B inclusions fixed and stained slide are merely "stopped of virus origin. action" of a process which progresses from At any time during the course of in­ an early to a late stage. In addition, it clusion formation steps leading to cell should be kept in mind that these series death may be superimposed upon inclu­ of events may be cut short at any time be­ sion bearing cells. One can summarize the cause superimposed upon them there may principal steps in autolysis as follows: 1. be other events which lead to death of the A change in the chromatin to make more cell before the inclusion body series has than normal amounts of oxychromatin vis­ had time to be completed. ible and a tendency for the chromatin granules to show a range in size greater BIBLIOGRAPHY than normal. 2. Frequently a slight lique­ 1. Cowdry, Edmund V. 1922. The supravital stain­ ing of vaccine bodies. J. Exper. Med. 36:667-684. faction may occur so that the nuclear sap 2. Woodruff, C. Eugene, and Goodpasture, Ernest W. 1929. The infectivity of isolated inclusion bodies shows a slight chromatin reaction and of fowl-pox. Am. J. Path. 5:1-9. 3. Goodpasture, E. W. and Woodruff, C. E. 1931. A sometimes accompanying this there is comparison of the inclusion bodies of fowl-pox and molluscum contagiosum. Am. J. Path. 7:1-7. slight hypertrophy of the nucleus. 3. A 4. Bland, J. O. W. and Robinow. C. F. 1939. The inelusion bodies of vaccinia and their relationship to coalescence of basophilic granules, de­ the elementary bodies studied in cultures of the rab­ struction of the linin framework except bits' cornea. J. Path. and Bact. 48:381-403. 5. Ludford, R. J .• and Findlay, G. M. 1926. The for a few strands and shrinkage of the nu­ ultra-microscopic viruses: II. The cytology of fowl­ pox. Brit. J. Exper. Path. 7 :256-264. clear membrane. 4. A further shrinkage 6. Cowdry, E. V. 1934. The problems of intranu­ clear inclusions in virus diseases. Arch. Path. 18 :527- of the nuclear membrane which draws the 542. 7. Lucas, Alfred M. 1940. The cytology of fox ence­ chromatin masses together, the nuclear phelitis and the effects of centrifugation upon the intranuclear inclusions. Am. J. Path. 16:739-760. sap disappears and a pycnotic nucleus is 8. Cowdry, E. V., Lucas, Alfred M., and Fox, Her­ the end result. This series of reaction is bert. 1935. Distribution of nuclear inclusions in wild animals. Am. J. Path. 11 :237-252. reversible in its early stages. 9. Green, R. G. 1936. Purification of fox encephali­ tis virus by trypic digestion. Jour. Bact. 32:120. Sometimes the inclusion bodies in two 10. Lucas, Alfred M., and Herrmann, Walter W. 1935. Effect of centrfugation on herpetic intranuclear inclu­ closely related diseases are similar but sions with a note on cytoplasmic inclusions of unknown origin in thEe rabbit cornea. Am. J. Path. 11 :969-976. use can be made of the fact that their 11. Nicolau, S., and Kopciowska. L. 1938. La mor­ phologie de l'inframicrobe herpetique dans Ie tissu occurrence is specific for certain tissues des animaux infectes experimentalement et Ie as has been done by Green and Evans in mechanisme de la formation des inclusions qu'il en­ gendre dans les cellules. Ann. Inst. Pasteur. 60:401-431. the separation of fox excephalitis and ca­ 12, Rosenbusch, Carlos T., and Lucas, Alfred M. 1939. Studies on the pathogenicity and cytological re­ nine distemper. Woodruff (22) differen­ actions of the submaxillary gland virus of the guinea pig. Am. J. Path. 15:303-338. tiated vaccinia virus and fowl pox in the ,3, Birch, Frank and Lucas, Alfred M. Centrifuga­ tion of intranuclear inclusions produced by aluminum same animal on a cytological basis. oxide. Work in progress. 14. Andrewes, C. H. 1930. Immunity to the saliv­ To safe-guard against over confidence ary virus of guinea pigs studied in the living animnl, and in tissue culture. Brit. J. Exp. Path. 11 :23-34. in the diagnostic value of all inclusion 15. Olitsky, Peter K., and Harford, Carl G. 1937. bodies, it is well to keep in mind the work Intranuclear inclusion bodies in the tissue reactions produced by injections of certain foreign substances. of Wolf and Orton (23) on the inclusion Am. J. Path. 13:729-748. 16. Olitsky, Peter K., and Harford, Carl G. 1938. bodies of poliomyelitis in which they found Further observations on intranuclear inclusions pro­ duced by non-virus materials. Proc. Soc. Exper. BioI. what cytologically appeared to be identi·· and Med. 38 :92-94. .. 17. Luger, A., and Lauda, E. 1926. Uber oxychro­ cal bodies in gliomas and diseases matische Veriinderungen am Zellkern (auf Grund von Untersuchungen von Herpes simplex, Zoster, Varizel­ in which the polio virus could play no len, Variola, und Karpfenpocke.) Ein Beitrag zui Ken­ part. The same attitude is probably cor­ ntnis und Wertung einschlussartiger Gebilde. Med. Klin, 22:415-417, 456-458, 493-497. rect in the evaluation of inclusions, both 18. Lee, Jack, 1933. Nuclear changes following intravenous injection of various solutions. Proc. Soc. nuclear and cytoplasmic, of equine en­ Exper. BioI. and Med. 31 :383-385. 19. Blackman, S. S. Jr. 1936. Intranuclear inclu­ cephalomyelitis. sion bodies in the kidney and liver caused by lead poisoning. Bull. John Hopkins Hosp. 58:384-403. In conclusion it is apparent that when 20. Belt. Thomas H. 1939. Liver necrosis following burns simUlating the lesions of yellow fever. J. Path. we have learned the real nature of all the and Bact. 48:493-498. 21. Underhill, F. P., Kapsinow, R. and Fisk, M. E. cellular bodies associated with virus dis­ 1930. Studies on the mechanism of water exchange eases, then their use for diagnostic pur­ in the animal organism, I. The nature and effects of superficial burns, Am. J. Physiol. 95 :302-314. poses will be greatly enhanced. Until that 22. Woodruff, C. E. 1930. A comparison of fow!­ pox and vaccinia in the chick with especial reference time comes, however, inclusions should to the virus bodies. Am. J. Path. 6:169-173. 23. Wolf, Abner, and Orton, Samuel F. 1932. The be studied with consciousness on the part occurrence of intranuclear inclusions in hUlnan nerve cells in a variety of diseases. Bull. Neural Inst, N. Y. of the observer that the inclusions on a 2:194-209, 46 The Veterinary Student