Rat virus infection of megakaryocytes: A factor in hemorrhagic encophalopathy?

EXPERIMENTAL

Rat

AND

MOLECULAR

Virus

PATHOLOGY

Infection

of Megakaryocytes:

Hemorrhagic GEORGE MARGOLIS, Departments

16, 326-340 (1972)

A Factor

in

Encephalopathy?’ AND LAWRENCE

KILHAM

and Microbiology, Dartmouth Hanover, New Hampshire 03756

of Pathology

Medical

Smchool,

Received December 8, 1971 Multiple contributory factors have been postulated in the pathogenesis of hemorrhagic encephalopathy of the rat, an entity induced by rat virus infection of sucklings. These include: (1) the direct attack of virus upon vascular endothelium with resultant thrombotic and hemorrhagic effects; (2) the triggering of consumptive coagulopathy by the vascular injury; and (3) the production of a deficiency coagulopathy secondary to viral hepatitis. This report describes a new feature of rat virus infection, the involvement of the pool of developing megakaryocytes. This cellular lesion, although unsupported by evidence of depressed platelet levels, or disturbed platelet function, suggests that a direct viral attack upon platelet-forming elements may be an additional contributory factor in the pathogenesis of hemorrhagic encephalopathy. The complex of pathogenetic factors for hemorrhagic encephalopathy of the rat indicates that this entity offers an excellent experimental model for study of the general problem of the pathophysiology of disturbances of blood coagulation in virus infections.

Viral hemorrhagic encephalopathy of the rat, a reproducible manifestation of rat virus diseaseof sucklings (Cole et al., 1970; Nathenson et al., 1970) has been suggested to be the resultant of the concurrence of three pathogenetic factors, namely: (1) the viral attack upon the proliferating vascular bed of the developing organism, (2) the induction of a consumptive coagulopathy secondary to vascular injury, and (3) the production of a deficiency of clotting factors secondary to the viral attack upon hepatic parenchyma (Margolis and Kilham, 1970). The present report provides evidence that still one more causative factor may, theoretically, be operative, i.e., an interaction of virus with the pool of developing megakaryocytes. MATERIALS

AND METHODS

This study consisted of a review of tissues made available from earlier investigations of parvovirus disease (Kilham and Margolis, 1965; Margolis et al., 1968; Margolis and Kilham, 1970). Where the foregoing reports had concentrated upon central nervous system manifestations of the viral infection the present paper is focused upon changes involving the pool of developing megakaryocytes in the spleen, bone marrow, and liver. Details of the experimental procedures from 1 Presented at AAPB Meeting, March 7-9, 1971, Montreal, Canada. This work was supported in part by U.S. Public Health Service Grants NB-05545, HD-03298, CA-06010, and l-KG-CA 22,652. Copyright

Q 1972 by Academic

Press,

Inc.

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VIRUS

INFECTION

OF

MEGAKARYOCYTES

327

which these source materials were derived are provided in these foregoing reports and thus are outlined but briefly here. Nine strains of rat virus were used including RV12, RV13, RV171, RV308, SREV, HER, Zhadenov, OLV, and HHP. Newborn to 5-day-old animals were inoculated by either intracranial or intraperitoneal routes, or by both routes, and infections were terminated from 1-21 days, usually between 3 and 13 days. The exsanguination perfusion fixation method of Malm (1962) was used to produce optimal preparation of the central nervous system, and extraneural tissues were immersion-fixed in Bouin’s solution. Unfortunately, the former method adversely affected the quality of preparation of extraneural tissues, particularly the spleen and bone marrow. Unless the spleen was removed beforehand, it was often unsuitable for critical study. Acceptable preparations of vertebral bone marrow were obtained only when the descending aorta had been clamped during perfusion. All tissues were carried through paraffin embedding, sectioned at 5-6 p and stained with hematoxylin-eosin. Characteristic intranuclear inclusions were used as indicators of viral infection. Of extraneural tissues from 232 infected rat sucklings satisfactory preparations were

FIG. 1. Two features of splenic megakaryocytopoiesis. The megakaryocyte to the left has 16 nuclei in or near the focal plane of the section. To the right a megakaryocyte undergoing nuclear division is illustrated. This replication without cytoplasmic division results in progressive polyploidy and increased numbers of nuclei. Kitten 14 days postinoculation. X875. All photomicrographs have been made from 5- to 6-p paraffin sections of Bouin’s fixed splenic &sues stained with hematoxylin and eosin.

328

MARGOLIS

AND KILHAM

available from approximately one-quarter. Six infected kittens were also studied; material from four of these was satisfactorily prepared. Selection of tissues for study was guided by the observation of Marien and McFadden (1968) that the spleen rather than other hematopoietic organs is the major locus of megakaryocytopoiesis in the suckling rat. Their studies indicated a dominance of this activity in the liver in utero. Late in gestation megakaryocytopoiesis became active in the spleen and this organ superceded the liver in this function within a few days after birth. On the other hand, the bone marrow did not become a significant contributor to this cell pool until about 40 days postnatally. Other aspects of these studies, including virologic procedures and details of the neurologic manifestations of virus infection are reported elsewhere (Kilham and Margolis, 1965; Kilham and Margolis, 1966; Margolis et al., 1968; Margolis and Kilham, 1970). RESULTS All of the animals used in this study exhibited severe generalized infections, in which hemorrhagic encephalopathy (Margolis and Kilham, 1970) and/or in-

FIG. 2. Two normal-appearing megakaryoblasts and one parasitized megakaryoblast. These cells are less mature than those in Fig. 1. the nucleus of one cell showing only early lobulation and polyploidy. The infected cell has five involved nuclei in the plane of focus. Here the inclusions are featured by condensation, drawing away from the nuclear membrane, and halo formation. Rat 10 days postinoculation. X1250.

RAT

VIRUS

INFECTION

OF

MEGAKARYOCYTES

329

volvement of the cerebellar external germinal layer (Kilham and Margolis, 1966) as well as hepatitis (Margolis, Kilham, and Ruffolo, 1968) were the outstanding features. Involvement of the megakaryocyte pool was readily observable only in the spleen. Fifteen of 75 infected rats and two of four infected kittens whose somatic organs were considered suitable for histologic study had specific splenic changes. In these only a small proportion of the splenic megakaryocyte series, never over 20%, was affected. Thus, in the face of the severe generalized infection, involvement of megakaryocytes was considered to contribute only in a modest way to the total disease picture. The inclusion-body phase of infection in the spleen was observed between 4 and 14 days, paralleling the active stage of the infectious process in other tissues. During the suckling stage in both rats and kittens mitotic activity was prominent in the megakaryocyte series (Figs. 1 and 2.1, accompanied by a progressive increase in ploidy, from morphologically elusive stem cells with single nuclei through typical large multinucleated megakaryocytes. Of interest and significance, the viral attack was concentrated upon apparent precursor cells (Fig. 3) and megakaryoblasts (Figs. 4 and 5) rather than upon mature mega-

FIG. 3. Multiple stem cells (arrows), considered on the basis of comparative studies to be megakaryoblasts, showing early inclusion-body stage of rat virus infection. These inclusions at this stage are eosinophilic, with variably discrete borders, are surrounded by a halo of lucent nucleoplasm, and are clearly separable from nucleoli. Kitten 14 days postinoculation. x 1375.

330

MARGOLIS

AND KILHAM

FIG. 4. Virus-infected megakaryoblast from same animal from which Fig. 3 was made. Here the nucleus is large and has an early polylobular pattern. The inclusion again is eosinophilic, has ill-defined borders, is distinctly separable from the nucleolus, and has a surrounding halo of lucent nucleoplasm. X2150. karyocytes. A characteristic feature of the infection was the uniformity of the appearance of inclusions within individual megakaryocytes. Here, whether segmentation into separate nuclei had occurred (Figs. 2, 5, 10-12) or whether a continuous polylobulated nuclear mass was present (Figs. 4, 6-9) all inclusions within a parasitized cell had an identical appearance. This feature was indicative of a synchrony of viral attack upon sites of synchronous synthesis of DNA. The earliest evidence of inclusion formation within this cell series was the appearance of an amorphous slightly eosinophilic material, generally centrally in the nucleus, independent, of the nucleolus and standing out against the otherwise lucent nucleoplasm (Figs. 3 and 10). As these inclusions enlarged and increased in density the nucleoplasm became even more lucent and the nuclear envelope increased in thickness and density, creating a prominent halo effect (Fig. 4). In some involved cells the inclusions were spread diffusely throughout the nucleoplasm, filling the nuclei completely and uniformly; here surrounding halos were virtually absent or inconspicuous, even in the presence of thickened nuclear envelopes (Figs. 6-9). At a more advanced stage in the progressive sequence these inclusions appeared more compact and retracted from the nuclear mem-

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OF MEGAKARYOCYTES

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Fra. 5. Infected megakaryoblast with four inclusion-bearing nuclei in the plane of focus. This cell is more mature and differentiated than involved cells in Figs, 2 and 3. The inclusions are amphophilic and are interpreted as having retracted from the nuclear margins. Rat 12 days postinoculation. X1650.

brane, producing surrounding halos with sharply defined borders (Figs. 2, 5, and 12). One other late stage was the appearance of multiple vacuoles within and around otherwise characterist,ic intranuclear inclusions (Figs. 10-12). Such moth-eaten cells have not previously been described in parvovirus infection. Even at these advanced stages of inclusion-body formation the cytoplasm of the parasitized cells presented no features of cell injury. An occasional circulating inclusion bearing megakaryocyte floating free within a splenic venule was encountered (Figs. 8 and 9). Ultimately death and lysis of the affected megakaryocytes occurred. DISCUSSION These observations of the interaction of virus and megakaryocytes, without further supporting data, cannot be offered as firm evidence of a new contributory factor to the pathogenesis of hemorrhagic encephalopathy induced by rat virus. Confirmatory data would require the demonstration of disturbed platelet functions, depression of platelet levels, or alterations in platelet-associated coagulation factors. They do, however, invite speculation about the possibility and point

MARGOLIS

332

AND

KILHAM

FIG. 6. Two infected cells in the megakaryocyte series. Both in the mononuclear cell (lower right) and the cell with a complex polylobulated nucleus inclusions are distinct from nucleoli. The inclusions in the latter cell are eosinophilic and loose-textured, features suggestive of an early stage of development. Kitten 14 days postinoculation. X1800.

to the need for further study of this relationship. For still other reasons these observations have intrinsic interest. Notably, they have provided the opportunity and the stimulus for presentation of an overview of the pathophysiology of virus-induced hemorrhagic tendencies, a subject made timely by the press of new discoveries

in t’his field.

Moreover,

these findings

furnish

an excellent

example

of

the dependent relationship between parvoviruses and replicating cells. Our earlier studies of parvovirus infections, while suggestive that hemorrhagic encephalopathy might be the direct result of the viral attack of vascular endothelium, brought out other possible pathogenetic factors (Margolis and Kilham, 1970). Among these was a deficiency coagulopathy secondary to hepatic involvement (Margolis, Kilham, and Ruffolo, 1968). Moreover, the possibility of a consumptive coagulopathy triggered by the endothelial injury, as suggested by McKay and Margaretten (1967) had to be considered. Recent ultrastructural studies by Baringer and associates (1971) have demonstrated perivascular cerebral bleeding in the presence of intact endothelium, again suggesting a more complex pathogenesis. Examples of the complex pathophysiology of hemorrhagic states associated with virus infections, and the incomplete status of our under-

RAT

VIRUS

INFECTION

OF

MEGAKARYOCYTES

FIG. 7. Infected megakaryoblast exhibiting late stage of inclusion-body formation. inclusion material is dense, amphophilic, and uniformly fills the polylobulated complex structure, except for a thin peripheral halo. Rat 10 days postinoculation. X2250.

333

The nuclear

standing of the pathogenesis of these processes are cited below, as an argument stressing the need for exploration of leads offered by new observations such as those reported presently. Recent studies of the pathogensis of dengue hemorrhagic fever illustrate the role of altered immunity in the response to a virus which attacks vascular endothelium (Halstead et nl., 1970; Halstead and Simasthien, 1970; Nisalak et al., 1970; Halstead et nl., 1970; Fischer and Halstead, 1970; Halstead, 1970). Here, the more benign course of the initial infection, “normal dengue,” is condisease occurring in trasted with the ‘(altered dengue,” the severe hemorrhagic reinfected individuals. In primary infections viral multiplication occurs in vascular endothelium, among other sites, and virus parasitized cells are probably eliminated either by virus-induced cytolysis or by delayed hypersensitivity. In “altered dengue” it is postulated that (Halstead, 1970) : “Two independent antigen-antibody reactions may contribute to the alteration of infection responses. Serum antibody (probably IgG), which complexes free virus and complement, may aggregate blood platelets and result in thrombocytopenia without shock. Increased capillary fragility is caused by an immune mechanism not measured by conventional serologic techniques. Alternate hypotheses

334

MARGOLIS

AND KILHAM

FIG. 8. Shows three inclusion-bearing cells (A, B, C). A, with a simple rounded nucleus, and B with a polylobulated nucleus float free in the lumen of a venule. Rat 12 days postinoculation. X900.

of the mechanism of capillary damage in both primary and secondary infection DHF are proposed: (1) Preinfection dengue antibody . . . complexes with virus produced during the initial stages of infection. These complexes stimulate anticomplex antibody. . . . As infection progresses, complexes and anticomplex antibody increase to critical cytotoxic levels. (2) Preinfection antibody . . . promotes a destructive acceleration of the delayed hypersensitivity response.” Although the hemostatic defects in dengue hemorrhagic fever have been postulated to be the result of triggering of intravascular coagulation (McKay and Margaretten, 1967; Fresh et al., 1969) the infrequency of disturbances of clotting factors argues against this interpretation. Congenital rubella represents an important example of neonatal thrombocytopenia and purpura of viral etiology, but the pathophysiology of these manifestations remains obscure. Bernard (1970) has summarized the state of our knowledge concerning this process as follows : “The role of rubella virus seems unquestionable, but the mechanism of its action remains unknown, and one can only cite the hypotheses proposed: (1) direct action of the virus on the megakaryocytes or on the platelets; (2) throm-

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OF MEGAKARYOCYTES

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FIG. 9. A segment of the field pictured in Fig. 7, showing the three parasitized cells. The inclusions in ;1 and B being dense and amphophilic, are considered to represent a later phase than those in C. While cells B and C are in the megakaryocyte series, the origin of A is uncertain. X 1800.

bocytolysis in the spleen; or (3) conflict between an antiplatelet antibody in the maternal serum and a virus-platelet complex in the infant.” Attal and Mozziconacci (1970), analyzing clinical features of rubella, emphasize that the direct action of virus upon megakaryocytes has not been proved. Svenningsen (1965) stated that it is seldom possible to reveal the presence of antiplatelet antibodies (one case in eight). Naeye (1965) and Plotkin et al. (1965) have presented evidence of a striking depression of cell division in rubella and suggestedthat this biologic action is an important factor in the pathogenesis of the spectrum of pathologic changes making up the rubella syndrome. Clearly, depression of megakaryocytopoiesis could be accounted for by this mechanism. Evidence of direct involvement of platelets has been obtained in studies with other viruses. Influenza virus has been observed to adsorb to, or become incorporated within platelets (Schulz and Landgraber, 1966) and the virus-platelet complex is eliminated from the circulation (Danon et al., 1959; Jerushalmy et al., 1963; Lu, 1958). Newcastle diseasevirus has been described as behaving like an ATPase, inducing viscous metamorphosis, release of platelet enzymes, and exhaustion of ATP (Cohen et al., 1966). Further studies of the interaction of

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MARGOLIS

AND KILHAM

FIG. 10. In the parasitized cell with two nuclei in the plane of section (upper right) the inclusion material is loose-textured, eosinophilic, and has irregular margins. In the infected cell with four nuclei in the plane of focus the inclusion material is dense, amphophilic, and has multiple vacuoles, particularly at its margins. Rat 10 days postinoculation, X2100.

platelets and viruses have been summarized by Maupin (1966). Bernard (1970) has reviewed the evidence suggesting a viral etiology for idiopathic thrombocytopenic purpura, and has concluded that the data are vague and insubstantial. Thus, it appears that hemostatic abnormalities associated with virus infections may be mediated by several mechanisms, including (1) direct viral attack upon vascular endothelium, (2) induction of consumptive coagulopathy secondary to vascular injury, (3) altered immune reactions, (4) direct viral attack upon platelets, (5) depression of hepatogenous coagulation factors, (6) depression of megakaryocytopoiesis as a feature of a generalized retardation of cell proliferation, and (7) direct viral attack upon the megakaryocyte pool. Other than the presently reported observations the best evidence for viral replication in megakaryocytes has been provided from studies of animals with virus-induced leukemia (DeHarven and Friend, 1958; Dalton et al., 1961; Dalton, 1962; Dalton and Moloney, 1962; Penelli et al., 1968). These investigations have demonstrated a persistent noncytolytic relationship between virus and cell. Viral particles are visualized by electron microscopy in the cytoplasm of mega-

MARGOLIS

FIG. 11. More advanced vacuolar cell in the megakaryocyte series. Rat

AND

337

KILHAM

changes in the nuclear 12 days postinoculation.

inclusion X1800.

material

of an infected

karyocytes of susceptible animals for long periods prior to leukemia manifestations and are found in high numbers during the course of the disease. Bernard (1970) has cited both the strengths and weaknesses of present approaches to study of leukemia viruses as follows: “Today we know how to culture all the leukemia viruses on embryonic or homologous hematopoietic cells, but study is impeded by the fact that none of them induces optically detectable lesions in the cells on which it grows.” Quite the opposite virus-cell relationship appears operative in the case of rat virus and megakaryocytes, i.e., a destructive viral action featured by a readily demonstrable inclusion-body phase. The intimate relationship between rat virus and the developing megakaryocyte provides further support for the concept of the special affinity of the parvoviruses and cells high in replicative activity and/or in DNA synthesis attending mitosis. The kinetics of megakaryocytopoiesis have been extensively studied. Megakaryocytes derived from a stem cell with a DNA value of 2N undergo increasing degrees of polyploidy, replication, and differentiation. Japa (1943) described five classesof megakaryocytes with 2, 4, 8, 16, and 32 nuclei, and considered to have developed by repeated normal mitotic division of their nuclei without cytoplasmic division. Garcia (1964) and Ode11et al. (1965) observed stepwise in-

338

FIG. 12. karyocyte,

MARGOLIS

Later stage of vacuolar change producing the typical moth-eaten

AND

KILHAM

in the nuclear inclusions appearance. Rat 10 days

of an infected postinoculation.

megaX2100.

creasesin DNA values to 4N, 8N, 16N, and 32N, by successive duplication of the original diploid amount of DNA. Ebbe (1968) from her own studies and analysis of literature concluded : “From a comparison of the results of measurement of megakaryocyte DNA (Ode11 et al., 1965) with those of labeling of megakaryocytes with 3HTdR (Feinendegen et al., 1962; Ebbe and Stohlman, 1965) it becomes apparent that there is a precursor pool in which polyploidy is achieved without mitosis.” About 25% of this cell compartment is in DNA synthesis at any one time and this activity appears to be synchronous in all nuclei of a cell (Feinendegen et al., 1962; Ebbe and Stohlman, 1965). Our own observations indicate that mitotic activity is readily observable in the susceptible megakaryocyte pool in the spleen of the suckling cat just as in the rat. In any event, the megakaryocyte series comprises a cell component which is engaged in active, synchronous DNA synthesis, and thus is an inviting target for a virus highly dependent upon host cell DNA metabolism (Margolis and Kilham, 1968; Margolis et aE., 1971). The synchrony in development of virus-induced inclusions in the individual nuclei of megakaryoblasts is completely consistent with and indeed supportive of this concept. Moreover, since replicative activity continues in megakaryocytes throughout the life span of the host, this cell series could, theoretically, be receptive to rat virus in the adult animal.

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