[CANCER RESEARCH 30, 1003—1010,April 19701

in this laboratory by strict brother-sister mating (7). Thesemice have been shown to have a very low incidence ofspontaneous leukemia (7). Electron microscopy of spleensand blood from uninfected mice never reveals any virus-likeparticles.

Virus. The origin of the MEV has been described (7). MEVused in the present experiments was obtained from cell-freespleen filtrates of the 23rd to 26th C3Hf/gs mouse passages.Throughout the passage history of MEV, virus potency andpathological manifestations of erythroblastosis have notdeviated from the original report (7). Animals were inoculated i.p. when I to 4 days old with 0.1 ml of the viruspreparation.

Red Cell Survival.@ â€C̃r-Labeled red cell survival studieswere carried out according to a modification of the methoddescribed by Donohue et a!. (6). Donor animals were lightlyanesthetized with ether, the brachial artery and axillary veinwere cut, and blood was collected in heparinized Pasteurpipets. Blood from several animals was pooled and centrifuged and the plasma and buffy coat were discarded. Theerythrocytes were washed once with 0.9% NaCI solution andresedimented. To each ml packed erythrocytes was added 1mCi sodium chromate-5 â€C̃r (New England Nuclear Corp.,Boston, Mass., 50 to 500 Ci/g, 1 mCi/ml in 0.9% NaClsolution). Donor cells were incubated at 37°for 45 mm,sedimented, washed twice with NaCl solution, and resuspended in NaCl solution to produce a 20% suspension. Eachrecipient animal received 0.5 ml of the suspension (0.1 mlpacked cells) i.p. Thirty-six hr elapsed before the firstsamples were drawn to permit complete absorption of redcells from the peritoneal cavity. Initial blood samples werecollected from the tail vein in 13-p1 capillary pipets. Subsequent samples were collected at 1 to 3-day intervals.Capillary pipets were stored in plastic vials and all samplesfrom 1 experiment were counted on the same day toeliminate the necessity for calculating radioactive decay.

5 , Cr Counting. Blood samples were counted at the

termination of an experiment in a Packard Auto.Gammaspectrometer. The first blood sample of a series containedgreater than 1000 cpm above a background of less than 20cpm or the results were not included in the experiment.Because of the great variability in the amount of radioactivity absorbed by individual animals, actual counts are notrecorded. Instead, the cpm recorded from the first sampleobtained from a mouse was arbitrarily considered as 100%,and all subsequent samples from that animal were recordedas percentages of the initial value.

Complete Blood Count. Hematocrits were determined inheparinized capillary tubes; white cells and platelets were

APRIL 1970 1003

Virus-induced Hemolytic Anemia in Mice'

Robert L. Wollmann, Emily J. Pang,Audrey E. Evans,and Werner H. KirstenDepartments of Pediatrics and Pathology, University of Chicago, Pritzker School of Medicine, Chicago, illinois 60637

SUMMARY

Mouse erythroblastosis virus induces splenomegaly, anemia,and thrombocytopenia in infected animals.@ ‘Cr-Labeledredcell survival studies and serial blood counts indicate that theanemia is due to red cell destruction and the splenomegaly isdue to a compensatory hyperplasia of erythrocytic pre.cursors. Splenectomy prevents the secondary reticulocytosis,but it does not prevent the red cell destruction. Electronmicroscopy of peripheral blood from mice infected withmouse eiythroblastosis virus demonstrates budding virusparticles at platelet and reticulocyte cell surfaces.

INTRODUCTION

Mice inoculated with MEV2 shortly after birth developsplenomegaly and a rapidly progressive anemia from whichthey die within 9 weeks (7). Histologically, the enlargedspleen and bone marrow contain masses of proliferating redcell precursors, predominantly basophilic erythroblasts,but all stages of maturation are present. The possibility wasconsidered that the primary disease induced by MEV is anerythroleukemia. The anemia in this case would be due toreplacement of normal erythropoietic tissue by malignanterythroblasts. However, the erythroblasts seen in this diseaseare indistinguishable from erythroblasts in normal controlmice and are not transplantable to syngeneic hosts. It wasalso considered that MEV has a direct effect on red cells,leading to hemolysis, as it has been reported for Friendleukemia (1).

In the present study, the nature of the anemia in murineerythroblastosis is examined. Serial hematological profiles ofMEV-infected mice and survival times of red cells labeledwith@@ Cr were determined in normal and MEV-infectedrecipients. The results indicate that MEV induces ahemolytic anemia and that the erythroblastic proliferation isa compensatory mechanism secondary to hemolysis.

MATERIALS AND METHODS

Mice. The C3Hf/gs mice used in these experiments wereentirely from an inbred colony maintained for over 8 years

â€T̃his work was supported by funds from the Leukemia Research

F@unthtion.The abbreviation used is: MEV, murine erythroblastosis virus.

Received August 4, 1969; accepted October 1, 1969.

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Wollmann, Pang, Evans, and Kirsten

counted directly with Unopettes (Becton, Dickinson and Co.,Rutherford, N. J.).

Reticulocytes were counted according to the brilliant cresylblue method. Bilirubin levels were determined on plasmaobtained from microhematocrit tubes by the method ofLeffler (8). With this method, turbid plasma will yield falselyhigh readings; therefore, any grossly turbid plasma wasdiscarded.

Splenectomy. At 24 to 48 hr after birth, the mice wereplaced in a deep freezer at 0°. When respiration andmovement had ceased, the animal was removed and thespleen was exposed in the left flank by an incision throughthe skin and peritoneum directly over the long axis of thespleen. The spleen was protruded by gentle pressure anddetached from its vessels and connective tissue. Care wastaken not to injure the pancreas. The incision was suturedwith silk. Virus was inoculated 24 hr after splenectomy, asdescribed above. All animals were autopsied at the end of anexperiment to ensure that splenectomy was complete.

Electron Microscopy. Blood from anemic animals wascollected in heparinized microhematocrit tubes, washed in0.9% NaCl solution, and pelleted at 3000 rpm for 10 mm.The 0.9% NaCI solution was replaced with cold 2% 0504 incollidine buffer, pH 7.4, and the pellet was fixed for 1 hr at4°. After fixation, the cell pellet was dehydrated andembedded in Epon, and sections were viewed in an RCAEMU 3D electron microscope. Blood from uninfectedcontrols was similarly treated.

RESULTS

Preliminary studies of C3Hf/gs mice inoculated as newbornswith MEV suggested that the cause of death at about 8weeks after virus inoculation was most probably due to asevere anemia (7). We therefore determined several hematological paramete@s during the progressive course of erythroblastosis. Twenty-five newborn C3Hf/gs mice were inoculatedwith MEV; 15 mice were set aside as noninoculated controls.Blood was collected from the retroorbital plexus intomicrohematocrit tubes at weekly intervals beginning at 3weeks of age. Hematocrits, reticulocyte, platelet and whitecell counts, and bilirubin content were determined for eachsample (Chart 1).

The MEV-infected mice showed rapidly falling hematocritsand rising reticulocyte counts, beginning at 3 weeks aftervirus inoculation. The anemia was accompanied by athrombocytopenia with platelet counts of less than 50,000during the last week of life. However, at autopsy, hemor.rhagic lesions were not seen, even in animals with plateletlevels that had fallen before death to less than 3% ofcontrols. Between the 4th and 6th weeks after virus inoculation, the hematocrits had dropped to 20%, but the stools ofthe animals were negative for hemoglobin when tested withbenzidine. Further evidence of red cell destruction was therise in serum bilirubin levels that accompanied the fall inhematocrit (Chart 2).

These results indicated that MEV induced a profoundanemia beginning about the 3rd week after virus inoculation.Red cell survival studies were undertaken to determine the

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Chart 1. Composite recording of hematological parameters measuredin normal and MEV-infected mice. The broken line in each graphconnects the average values obtained in each case from 25MEV-infected mice and the solid line connects the average values from15 littermate controls. The vertical line through each point representsthe standard deviation from the average. Upper left, state of red cellvolume (hematocrit) at various times after birth; upper right,percentage of reticulocytes; lower left, white cell count (per cu mm);lower right, platelet count (per cu mm).

Chart 2. Serum biirubin levels in MEV-infected and control mice. Thebroken line in each graph connects the average values obtained in eachcase from over 15 MEV-infectedmice, and the solid line connects theaverage values obtained from over 15 littermate controls. The verticalline through each point represents the standard deviation from theaverage. The upper graph was produced with values obtained from theserum of totally splenectomized mice of Chart 6. The lower graph wasproduced with values obtained from the serum of nonsplenectomizedmiceofChart 1.

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Chart 4. Survival of@@ Cr-labeled red cells from MEV-infected donorsin 10 normal and 10 MEV-infectedrecipients. Each point represents theaverageand the vertical line rerresents the standard deviation from theaverage of the percentage of â€C̃r counts remaining in the peripheralblood of the animals at each specified time. This value is derived bycomparing the@@ Cr counts from a 13-@tlaliquot of blood obtained on agiven day, with the counts from an aliquot obtained 24 to 48 hr aftertransfusion of@ â€C̃r-tagged erythrocytes (Day 0) and arbitrarilyconsidered 100%. The broken line connects values derived fromMEV-infected mice and the solid line connects values from normallittermate controls.

20

Virus-induced Hemolytic Anemia in Mice

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nature of the anemia. For this purpose, the survivals of5 , Cr-tagged red cells from normal animals were compared

between groups of normal and MEV-infected C3Hf/gs mice(Chart 3). In addition, the survival of red cells from MEVinfected anemic animals was determined (Chart 4).

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Chart 3. Survival of@@ Cr-labeled red cells from normal donors in 10normal and 10 MEV-infected recipients. Each point represents theaverage and the vertical line rerresents the standard deviation from theaverage of the percentage of@ Cr counts remaining in the peripheralblood of the animals at each specified time. This value is derived bycomparing the@@ Cr counts from a 13-gzlaliquot of blood obtained on agiven day, with the counts from an aliquot obtained 24 to 48 hr aftertransfusion of@@ Cr-tagged erythrocytes (Day 0) and arbitrarilyconsidered 100%. The broken line connects values derived fromMEV-infected mice and the solid line connects values from normallittermate controls.

The survival rate of normal cells in MEV-infected animals(5 1 Cr t½ 1 .7 ± 0.5 days) was markedly decreased

compared to the 50% survival of the same cells in normalcontrol animals (5 1Cr t½ 16.3 ±3.3 days) (Chart 3).

The survival rate of red cells from MEV-infected anemicdonors was compared in normal and MEV-infected recipients. Here, the life-span of the red cells was also markedlyshortened when inoculated into infected animals; (5@ Cr t½= 2.1 ± 0.7 days as compared with the survival of the same

cells in normal recipients,@ â€C̃r t½ 11.2 ± 1.8 days)(Chart 4).

In addition to the obviously shortened survival when5 1Cr-tagged red cells were inoculated back into infected

recipients, the life-span of cells from infected anemic animals(5 1 Cr t½ 1 1 .2 ± 1 .8 days) (Chart 5) was significantly

shorter in normal recipients, p < 0.001 , than the life-span ofcells from control animals (5@ Cr t½ 16.1 ±I .4 days).

From these results, it appeared that a significant component of hemolysis must have been due to an intrinsic

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Chart 5. Comparison of the survival in normal recipients of5 â€C̃r-labeled red cells from normal donors (solid line) with@@ Cr-labeled

red cells from MEV-infecteddonors (broken line).

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animals (5 1Cr t½ 2.9 ±1.7 days) was significantly (p <-- .. 0.05) longer than the survival of normal red cells in MEV

3 5 -- age (wk) infected mice of the same age that were not splenectomized

(51Cr t½ 1.7 ±0.5 days).Autopsy findings from mice with erythroblastosis have

been reported (7). The term erythroblastosis was chosenbecause it best described the erythroproliferative process inthe spleen, liver, and bone marrow. Multiple clusters of redcell precursors are present in hepatic or splenic sinusoids andthe marrow spaces. About 75% of the spleen or bonemarrow population belong to the erythrocytic series. Basophilic erythroblasts are identified as the most common cellform within the red cell series (7). Thus, the cellularresponse of the disease is confined to organs that normally

_____________ _____ produce red cells in mice, while other organs reflect the:;‘;‘ ageColi) severe anemia, erythrophagocytosis, and hemosiderosis, as

evidenced in the liver, spleen, and lymph nodes.Histological material was avaUable from splenectomized mice

and mice which had inadvertently been partially splenectomized. A greatly accentuated hemosiderosis characterized thesplenectomized mice with erythroblastosis. Hemosiderosis(Fig. 3) and erythrophagocytosis (Figs. I and 2) wereespecially severe in mesenteric and parathymic lymph nodes.Prussian blue stains did not disclose hemosiderin in pancreas,myocardium, striated muscle, lungs, or salivary glands. The

5 7 age(wk)

Wollmann, Pang, Evans, and Kirsten

defect in the red cells from MEV-infected mice. However,the major component of hemolysis must have been due tohost factors of infected animals reacting against donor cells,since red cells from both normal and MEV-infected micewere rapidly destroyed when inoculated back into MEVinfected animals.

Since splenomegaly was the main gross clinical finding ininfected animals, the primary defect leading to anemiaconceivably could have been due to excessive destruction ofotherwise normal cells by an abnormally functioningreticuloendothelial system. Previous morphological findingsindicated that the splenomegaly of mice with erythroblastosis was caused by erythroblastic proliferation, ratherthan erythrocyte sequestration. We therefore compared redcell survival and the other hematological parameters insplenectomized and nonsplenectomized mice.

No significant differences were found in blood countsbetween normal splenectomized mice and nonsplenectomizedmice of the same age, except for a constantly elevated whitecell count in splenectomized mice, (Chart 6). The MEVinfected splenectomized mice failed to develop a reticulocytosis and increased bilirubin levels (Chart 2) despite thepresence of a severe anemia. In addition, the@ â€C̃r survivalrate of normal cells in splenectomized uninfected mice didnot differ from the@ â€C̃r survival rate of normal red cells innonsplenectomized mice. Chart 7 demonstrates that thesurvival of normal red cells in splenectomized MEV-infected

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Chart 6. Composite recording of hematological parameters measuredin splenectomized normal and splenectomized MEV-infected mice. Thebroken line in each graph connects the average values obtained in eachcase from 18 MEV-infectedmice and the solid line connects the averagevalues from 15 littermate controls. The verticalline through eachpointrepresents the standard deviation from the average. Upper left, state ofred cell volume (hematocrit) at various times after birth; upper right,percentage of reticulocytes; lower left, white cell count (per cu mm);lower right, platelet count (per cu mm).

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Chart 7. Survival of@@ Cr-labeled red cells from normal donors in 12splenectomized normal recipients and 10 splenectomized MEV-infectedrecipients. Each point represents the average and the vertical linerepresents the standard deviation from the average of the percentage ofS Cr counts remaining in the peripheral blood of the animals at each

specifiedtime. This value is derivedby comparing the@ â€C̃r counts froma 13-@tlaliquot of blood obtained on a given day with the counts froman aliquot obtained 24 to 48 hr after transfusion of@@ Cr-taggederythrocytes and arbitrarily considered 100%. The broken line connectsvalues derived from MEV-infected mice and the solid line connectsvaluesfrom normal littermate controls.

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Virus-induced Hemolytic Anemia in Mice

spleen fragment had enlarged considerably in partiallysplenectomized mice. Erythroblastic foci in the liver wereregularly found in the presence of an erythroblastotic spleenfragment but not without it. Erythroblasts and megakaryocytes were regularly found in lymph nodes of splenectomized mice.

The bone marrow of splenectomized and nonsplenectomized virus-inoculated mice was indistinguishable. Themarrow was hypercellular because of erythroid hyperplasia.However, megakaryocytes and myeloid cells were present asin uninoculated control mice. The marrow was the onlyorgan in which the erythroproliferation was evident irrespective of the presence or absence of the spleen.

From the preceding results, it appeared that, although thespleen played a significant role in the destruction of erythrocytes, it was by no means the primary cause of the anemia.Since the anemia was paralleled by a viremia (7), weconsidered the possibility that MEV interacted directly withred cells to cause their destruction. Blood smears did notsuggest agglutination of red cells in the peripheral blood ofmice with advanced erythroblastosis. To ascertain whetherthere was any direct physical interaction of red cells withthe virus, blood from anemic animals was centrifuged andthe resulting pellet was fixed and embedded for electronmicroscopy. As was demonstrated in Chart 1, a largemajority of the red cells in anemic animals at 6 weeks afterinoculation consisted of reticulocytes that still containedribosomes and occasional mitochondria. Mature virusparticles were not seen adherent to these red cells. However,several reticulocytes contained virus particles in the processof budding from the cell membranes (Figs. 4, 5, and 7). Inaddition, virus particles were also seen budding from plateletmembranes (Fig. 6). Examination of the buffy coat portionof the pellet failed to reveal budding virus at the surface ofany of the nucleated cells. Examination of blood fromuninoculated control animals did not reveal any virus partides. Electron microscopic examination of tissues fromerythroblastotic mice revealed virus particles budding fromthe cell membranes of megakaryocytes and erythroblasts (7).

DISCUSSION

In the original report on a virus-induced mouseerythroblastosis, we described splenomegaly as the first grossmanifestation of the disease (7). Splenomegaly coincided witha rapidly progressive anemia which eventually killed the host.Histologically, erythroblastosis was due to excessiveproliferation of red cell precursors in the spleen, as well as inother hemopoietic tissues. The erythroblastic proliferationappeared not to represent an autonomous neoplasm, sincespleen cell suspensions were not transplantable to adultsyngeneic recipients. Furthermore , normal maturation of redcell precursors was evident in tissues which are physiologicallyconcerned with erythropoiesis. We concluded that theerythroblastosis was, most likely, a reactive hyperplasiasecondary to red cell destruction (7).

The anemia from which MEV-infected mice die was causedby hemolysis, as demonstrated by a falling hematocrit, a risein reticulocyte count, and a rise in serum bilirubin levels at

about 3 weeks after MEV infection. Attempts were thereforemade to determine whether the hemolysis was due to anintrinsic defect in the erythrocytes of MEV-infected animalsor to an abnormally functioning reticuloendothelial system.In@@ Cr red cell survival studies we were unable todistinguish between these 2 destructive mechanisms.

The@ â€C̃r t½of red cells from MEV-infected mice wassignificantly shorter than the@@ Cr t½of red cells fromnormal mice in normal hosts ( I 1.2 ±I .8 days comparedwith 16.3 ±3.3). This would indicate that the red cells fromMEV-infected animals were defective compared with red cellsfrom normal animals, so that they were more rapidlyremoved from the circulation by a normally functioningreticuloendothelial system.

However, the life-span of@ â€C̃r-labeled red cells fromnormal animals was drastically shortened when inoculatedinto MEV-infected recipients. The@ â€C̃r t½ of normal redcells in MEV-infected mice was 1.7 ±0.5 days, as opposedto 16.3 ±3.3 days in normal hosts. These results indicatedthat the major defect responsible for the massive hemolysisin infected animals was extrinsic to the red cell.

Since splenomegaly accompanied the anemia, the questionarose as to whether the anemia was attributable to hypersplenism. The course of mouse erythroblastosis was thereforestudied in splenectomized mice.

Splenectomy had no effect on the survival of red cells fromnormal mice in uninfected hosts. In addition, removal of thespleen had little effect on the course of the disease andlongevity of a mouse subsequently inoculated with MEV.However, the@ â€C̃r t½of red cells from normal animals inMEV-infected recipients was slightly increased in splenectomized animals as opposed to animals with enlarged spleens,indicating that the spleen had a small part in removing redcells from the circulation. Whatever benefit splenectomyplayed in decreasing red cell destruction was apparentlyovershadowed by the loss of erythropoietic tissue. Splenectomized mice infected with MEV became anemic at about thesame time as nonsplenectomized animals but failed todevelop the reticulocytosis of intact animals. Splenectomy,in fact, did little to lengthen the life-span of red cells inMEV-infected animals. The@ â€C̃r t½ of red cells fromnormal donors was increased from 1.7 ±0.5 days to 2.9 ±1.7 days in splenectomized animals, which represented onlya minor reduction of red cell destruction, compared with aS 1 Cr t½ of I 6. 1 ± I .4 days in uninfected splenectomized

hosts.The results presented here are in general agreement with

those of Brodsky et al. (1) and Dennis and Brodsky (4), whoconcluded from ferrokinetic studies in Friend virus-infectedmice that the splenomegaly was in part a compensatoryresponse to hemolysis.

Electron microscopy offered some hints as to the possiblemechanism of red cell destruction in MEV infected mice.Virus production was demonstrated in megakaryocytes anderythroblasts of MEV-infected spleens (7). This phenomenonhas been described before for other mouse-leukemia viruses(2, 3, 9). In addition, as previously described (9), virusparticles were seen in the process of budding from reticulocytes and also from platelets of the peripheral blood. The 2

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Wollmann, Pang, Evans, and Kirsten

cell types that were most severely affected by MEV infectionwere the red cells and platelets. It is conceivable that virusparticles in the process of budding incorporate some antigenic components from the host cell membrane into thevirus envelope (5). The infected animal then makes antibodyagainst the entire spectrum of virus antigen, some of whichis derived from the host membrane. Antibody thus producedmay be capable of acting, not only against reticulocytes orplatelets with budding virus particles attached, but alsoagainst normal cells, thereby inducing the hemolytic anemiaand thrombocytopenia of mouse erythroblastosis. Immunological studies are now underway to determine whetherMEV-infected mice do indeed contain antibodies againstnormal red cells.

ACKNOWLEDGMENTS

We thank Mrs. Tina Raineri, Miss Carol Ganan, and Mrs. KatieBedford for excellent technical assistance and Mrs. Ethel Childressforpreparation of this manuscript.

REFERENCES

1. Brodsky, I., Dennis, L. H., and Kahn, S. B. Erythropoiesis inFriend Leukemia: Red Blood Cell Survival and Ferrokinetics.Cancer Res., 26: 1887—1892,1966.

2. Dalton, A. J. The Moloney Agent. In: A. J. Dalton and F.Haguenau (eds.), Tumors Induced by Viruses: UltrastructuralStudies, pp. 207—218.New York: Academic Press, Inc., 1962

3. De Harven, E. Ultrastructural Studies on Three Different Typesof Mouse Leukemia. In: A. J. Dalton and F. Haguenau (eds.),Tumors Induced by Viruses: Ultrastructural Studies, pp.183—206.New York: Academic Press, Inc., 1962

4. Dennis, L. H., and Brodsky, I. Thrombocytopenia Induced by theFriend Leukemia Virus. J. Nail. Cancer Inst., 35: 993—999,1965.

5. De The, G. Associations of Enzymes: Adenosinethphosphataseand Alkaline Phosphatase with the Virions of Murine Leukemias,NatI. Cancer Inst. Monograph, 22: 167—187,1966.

6. Donohue, D. M., Motuisky, A. G., Giblett, E. R., Pirzio-Biroli,G., Viranuvatti, V., and Finch, C. The Use of Chromium as aRed-Cell Tag. Brit. J. HaematoL, 1: 249—265, 1955.

7. Kirsten, W. H., Mayer, L. A., Wollsnann,R. L., and Pierce, M. I.Studies on a Murine Erythroblastosis Virus. J. Nati. Cancer Inst.,38: 117—139,1967.

8. Leffler, H. Microanalysis of Serum Bilirubin by Direct Spectrophotometry. In: F. Sunderman and F. Sunderman, Jr. (eds.), TheClinical Pathology of Infancy. Springfield, Ill.: Charles CThomas, Publisher, 1967.

9. Zajdela, F., Tambourin, P., Wendling, F., and Pierre, 0. Formation de Particules Virales sur Ia Membrane d'Erythrocytes deSouris Inject@es avec le Virus de Friend. Comp. Rend. Aced. Sci.,267: 2394—2396,1968.

Fig. 1. Mesenteric lymph node from a totally splenectomized MEV-infected mouse, demonstrating dilation of the sinusoids by phagocyticcells. X 175.

Fig. 2. Higher magnification of the same lymph node, demonstrating erythrophagocytosis by sinusoidal macrophages. X 640.Fig. 3. Prussian blue stain of the same lymph node, demonstrating hemosiderin deposits in sinusoidal macrophages. X 175.Figs. 4 and 5. Virus particle budding from reticulocyte membrane. X 35,000.Fig. 6. Virus particle budding from platelet membrane. X 33,600.Fig. 7. Virus particle budding from reticulocyte membrane. X 33,600.

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1970;30:1003-1010. Cancer Res   Robert L. Wollmann, Emily J. Pang, Audrey E. Evans, et al.   Virus-induced Hemolytic Anemia in Mice

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