(CANCER RESEARCH 27 Part 1, 322-332, February 1967]

Sites of Avian Leukosis Virus Multiplication inCongenitally Infected Chickens1

ROBERT M. DOUGHERTY AND HENRY S. Di STEFANO

Departments of Microbiology and Anatomy, Stale University of New York, Upstate Medical Center, Syracuse, New York

Summary

The distribution of virus in organs of chickens and chick embryos congenitally infected with avian leukosis viras (ALV) wasestablished by virus assay. The sites of virus multiplication incells of various tissues were determined by electron microscopy.Infectious virus was detected in all organs examined, and thehighest titers were found in liver and kidney. The adult femalereproductive system also contained large amounts of virus, whilelesser amounts were found in spleen, intestine, muscle, brain, andblood.

The presence of virus buds was used as evidence of virus multiplication in s]>ecific cells of various tissues and organs. Virusmultiplication was found to take place in cells derived from all 3embryonic germ layers, and virus buds were seen in cells of everytyi>e of tissue examined except nervous tissue.

Cells which supixjrted ALV multiplication included all 3 tyj)esof muscle, chondroblasts, fibroblasts, epidermal cells, liningepithelial cells of digestive organs, glandular epithelium of digestive organs and salivary glands, epithelial cells of kidney, cyto-reticular epithelium of thymus and bursa of Fabricius, endo-thelium, mesothelium, and primitive reticular cells of the spleen.

Introduction

Congenital transmission of avian leukosis virus (ALV) hasbeen established as an important mechanism for maintenance ofinfection under natural conditions (3,17). Studies of this phenomenon were carried out in a flock of chickens, all of which were con-genitally infected and transmitted virus to their progeny throughthe egg. It was shown that virus multiplied in the developing reproductive system of female embryos and in ovary and oviduct ofimmature and sexually mature hens. The quantity and locationof virus in the female reproductive system made it seem likelythat embryos became infected at a very early stage, ]>ossibly asa single cell (6, 7). Thus, i>otentially, all cells in a eongenitallyinfected chicken might themselves be infected with ALV. Thispaper reports results of a survey which was conducted to determine the extent of viras multiplication in chick embryos andchickens congenitally infected with ALV.

Materials and Methods

VIRUS.The strain of avian leukosis virus used in this study(ALV-F42) was isolated in tissue culture from the liver of a

1This work was supported by research grant No. GB 2129fromthe National Science Foundation and E328A from the AmericanCancer Society. Some of the equipment was purchased with fundsfrom NIH Grant 5TI GM 326.

Received July 18, 1966;accepted September 12, 1966.

chicken with a field case of lymphoid leukosis (visceral lympho-matosis). The original liver tissue was supplied by Dr. PeterBiggs, who independently isolated a strain of virus from the sameliver. The oncogenic spectrum in chickens of the F42 strain ofALV is similar to that of other field strains of viras isolated fromlymphoid leukosis (2), and its antigenic properties and interference spectrum place it in Group A of the classification schemeproi>osed by Vogt and Ishizaki (21).

VIRUSASSAYS.The COFAL (complement fixation for avianleukosis) test described by Sarma et al. (18) was ased as an endpoint for virus assays. Dilutions prepared from homogenates ofinfected tissues were inoculated onto chick fibroblast tissue cultures. The cells were subcultured 3 times and tested for the presence of COFAL antigen by a micro complement-fixation test (19).Antiserum for COFAL tests was obtained from Syrian hamsterswith tumors induced by the Schmidt-Ruppin strain of Rousvirus. Viras titers were expressed as the highest dilution (w/v) ofthe original tissue that gave a positive complement-fixation reaction after 3 passages in tissue culture. Tissue culture methodswere described in an earlier paper (9).

MICROSCOPY.Tissue samples were removed under anesthesiaand immediately fixed with a &'.v solution of glutaraldehyde in

McEwen salt buffer (pH 7.2), as before (6). No anesthesia wasused in removing tissue samples from embryos; ether was ased toanesthetize young chicks, while in older animals, Nembutal wasadministered i.v. After overnight fixation, the tissue sampleswere washed with half-strength McEwen buffer, treated for 1 hrwith a 1% solution of osmium tetroxide in McEwen buffer,washed again as before, dehydrated, and embedded in eitherEpon (15) or Maraglas (12, 20); some samples were embeddedin Maraglas using the procedure described by Erlandson (11).Thin sections were cut with glass knives asing an LKB Ultra-tome, double stained with uranyl acetate (22) and lead citrate(16), and examined with an RCA type EMU-3G electron microscope at an accelerating voltage of 100 kv.

SOURCK OF CONGENITALLY INFKCTKD CHICKENS. The initial

requirement of this investigation was a flock of viremic hens,congenitally infected with ALV, that regularly transmitted virusto their progeny through the egg. This was accomplished byrearing birds derived from embryos infected in the laboratory.Fertile eggs that were free of detectable infection with ALV (i.e.,"RIF-free") were purchased from SPAFAS, Inc., Norwich,

Connecticut. Embryos in the 13th day of development were infected i.v. with IO4infectious units of ALV and hatched. Surviv

ing birds were bled at the time of hatching and at 33 weeks and68 weeks of age, and the bloods were tested for ALV and forantibodies against an antigenically similar strain of Rous virus.

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Sites of ALV Multiplication

TABLE 1DISTRIBUTIONOFAVIANLEUKOSISVIRUSIN CONGENITALLYINFECTEDCHICKENSANDCHICKEMBRYOS

GENERATION"112233AGE42

weeks42weeks37weeks30weeks19-day

embryo19-day

embryoSEXFFFFFMVlïUS

TITER6 INTISSUEBloodIO5IO2IO5IO4NTNTLiverIO7IO5IO8IO6IO8IO5KidneyIO7IO6IO6IO7IO8IO5Spleen10«IO4IO6IO6IO8NTIntestineNTcNTNTNTIO510'Breast

muscleNTNTNTNTIO5IO4Brain10*<10'NTNTIO6IO5Gonad10«10»IO710sIO5IO2Oviduct10«IO3IO710sNTNT

" Generation: Ist-generation birds were infected in the laboratory as 13-day embryos. Subsequentgenerations were derived from the 1st generation.

bVirus titer is expressed as reciprocal of the highest dilution of tissue extract that gave a positiveCOFAL (complement fixation for avian leukosis) test after 3 passages in chick fibroblast tissue cultures.

c NT, not tested.

All of the birds that were tested were viremic throughout theexperiment, and none produced antibodies against the virus. Of48 birds that hatched and survived the 1st bleeding, 22 died ofhistologically confirmed visceral lymphomatosis, 16 were killedor died accidentally, and 3 died of unknown causes.

All hens that reached sexual maturity produced eongenitallyinfected embryos. A 2nd-generation, congenitally infected flockderived from these embryos eventually reached maturity, and a3rd-generation infected flock is being reared. A more detaileddescription of these flocks was given in another publication (7).

Results

A variety of organs from chick embryos and chickens that werecongenitally infected with ALV were examined for the presenceof infectious virus. The results of COFAL titrations of 10%tissue extracts, summarized in Table 1, showed that virus waswidely distributed. Liver and kidney consistently contained thelargest amounts of virus, but high titers were also obtained frommost other tissues. The female reproductive system was a richsource of virus, which confirms previous morphologic evidenceof prolific virus multiplication in these tissues (6, 7). Blood was arelatively poor source of virus compared with most other tissues.Since virus titers in most tissues were higher than virus titers inblood (brain was a notable exception), the amount of virus inmost organs could not be accounted for simply by contaminationwith infected blood. Either virus was concentrated from bloodby some mechanism or virus multiplication occurred in cellswithin the various organs. In view of this, morphologic evidenceof virus multiplication was sought, based on demonstration ofparticles in the process of budding. The detection of mature virusand budding forms, by electron microscopic examination of pancreas, was used to reinforce the virologie evidence of infection.The pancreas of all individuals used in this study contained largequantities of free virus particles and evidence of budding fromplasma membranes of acinar cells.

The results reported herein are from a survey of various tissuesand cell types associated with a variety of organs (externaloblique muscle, heart, embryonic limb bud, skin, pancreas,thymus, submandibular salivary gland, thyroid, duodenum,

cecum, rectum, liver, bursa of Fabricius, spleen, kidney, bonemarrow, cerebral cortex, spinal cord, and nerve of Rcmak).

Muscle

Examination of muscle from ALV-infected animals revealedthat all types of muscle cells supported virus growth. A virusparticle is seen budding at the surface of a skeletal muscle fiberin Fig. 1, a cardiac muscle fiber in Fig. 2, and a smooth musclecell in Fig. 3.

Cartilage

Cartilage, derived from an embryonic limb bud, was chosen asan example of a tissue associated with the skeletal system. Avirus particle growing at the plasma membrane of a chondroblastis seen in Fig. 4.

Connective Tissue

Connective tissue was a coni|x>nent part of most organs examined in this study. In each case virus budding from the surfaceof fibroblasts was a common occurrence. This is illustrated inFig. 5, in which virus particles are seen budding from fibroblastsin the pancreas.

Epithelium

This tissue is widely distributed throughout the entire organism. It forms the surface coating, lines the lumina of most hollowviscera, and comprises the parenchyma of all glands. In thisstudy, epithelial cells of the skin, kidney, lining of the gut andbile duct, and the parenchyma of pancreas, liver, intestinal glands,thyroid, bursa of Fabricius, thymus, and submandibular salivaryglands were examined for evidence of virus multiplication.

Although not similarly derived and therefore not epitheliumin the strict sense, we include endothelium and mesothelium inthis section on epithelium.

SURFACEEPITHELIUM.Examination of sections of skin showedevidence of virus multiplication in the squamous epithelial cellsof the Malpighian layer (Fig. 6), particularly in the stratumgerminativum and deep cells of the stratum spinosum as well as

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Robert Ai. Dougherty and Henry S. Di Stefano

in the fibroblasta associated with the dermis. No virus multiplication was observed in the more superficially located epidermalstrata.

LININGEPITHELIUM.Virus multiplication in lining epithelialcells of the oviduct has been rejxirted (6, 7). Growth of virus hasalso been observed in the lining epithelial cells of all organs of thedigestive system that were examined (duodenum, cecum, rectum,and interlobular bile ducts). An electron micrograph demonstrating a virus particle in the process of budding from a liningepithelial cell of the duodenum is seen in Fig. 7, and from a liningcell of an interlobular bile duct, in Fig. 8. The particle in Fig. 8was budding into the duct lumen, while that in Fig. 7 was buddingfrom the plasma membrane at the base of the cell. Budding frommucus-producing goblet cells of the intestinal lining was neverobserved.

GLANDULAREPITHELIUM..An earlier study (7) with hens thatwere congenitally infected with ALV showed that, in addition topancreatic acinar cells, the glandular epithelial cells of the oviductsupported virus growth. To ascertain whether other cells of thistype might also be involved in virus multiplication, glandularepithelial cells from a variety of organs were also examined andwidespread evidence of virus multiplication was noted, except forthe epithelial cells of the thyroid follicle. Virus buds were seen atthe surface of liver parenchymal cells (Fig. 9), epithelial cells ofglands in the intestinal mucosa (Fig. 10), and seromucous epithelial cells of the submandibular salivary glands (Fig. 11). Evidence of virus multiplication was also noted in epithelial cells ofthe kidney. Here, virus particles were seen budding from cells ofthe proximal (Fig. 12) and distal (Fig. 13) convoluted tubules andfrom cells of the collecting ducts (Fig. 14). Buds frequentlyentered the lumina of tubules and ducts. In the glomerulus, freevirus particles and virus budding into Bowman's space were ob

served frequently from the epithelial cells of the glomerulus. Abud at the surface of a capsular epithelial cell surrounding aglomerulus is seen in Fig. 15, while Fig. 16 demonstrates the samephenomenon at the surface of a podocyte; these particles are beingreleased into Bowman's space.

CYTORETICULAREPITHELIUM.This tissue makes up the fundamental stromal cell type characteristic of certain lymphoidorgans; free cells (i.e., lymphocytes) are found within a matrix ofthese cells. Evidence of virus multiplication was found in theepithelial cells, as well as in the free cells, of both bursa ofFabricius and thymus from birds that were congenitally infectedwith ALV. Fig. 17 demonstrates a virus particle in the process ofbudding from a cytoreticular cell in the bursa of Fabricius andbudding from a lymphocyte in the same organ is seen in Fig. 18.

ENDOTHELIUMANDMESOTHELIUM.During the course of thisinvestigation many imall blood vessels were encountered in thevarioas organs examined, and on occasions, sections of viscerathat were examined contained the serous membrane which surrounds the organ. Here, too, occasional budding of virus particleswas observed. This is shown in Fig. 19, where a virus particle isseen budding from an endothelial cell into the vessel lumen, andin Fig. 20, where virus is budding from a mesothelial cell of thevisceral peritoneum into the body cavity.

Reticular Tissue

This type of tissue is found in blood-forming organs such asspleen, bone marrow, and lymphoid organs. These are the fixed

cells and constitute the reserve or primitive reticular cells andthe phagocytic reticuloendothelial cells. The former develop intothe latter and into the various types of free cells found withinthese organs.

Virus particles were seen budding from primitive reticular cellsof the spleen (Fig. 21). Examination of the free cells in this organrevealed evidence of virus multiplication associated with lymphocytes similar to that observed above in the bursa of Fabricius(see Fig. 18).

In bone marrow, no evidence of virus multiplication was observed in either the reticular cells or any of the free cells of themyelogenous or erythropoietic series. In fact, after examiningmany sections only an occasional free virus particle was seen,despite the fact that many other tissues from the same chickensshowed abundance of virus particles. In 1 section, 2 virus particles were seen budding from a smooth muscle cell of a smallartery.

Nervous Tissue

Samples were taken of cerebral cortex, thoracic spinal cord,and nerve of Remak (an intestinal nerve comprising the sacralparasympathetic outflow). Careful and extensive examination ofthe nerve tissue that makes up these organs revealed an occasional free virus particle, but no evidence of virus multiplicationwas observed. This is consistent with the data in Table 1 andsuggests that infectious viras found in the brain may be carriedthere by way of the vascular system from some other site of multiplication.

Discussion

This investigation was provoked by the finding that germinalcells of hens congenitally infected with ALV are exiwsed to virusin the ovary, and the zygote is exjiosed to very large concentrations of virus in the oviduct prior to the 1st segmentation division (7). If the ovum or the zygote were infected and virus eitherintegrated into the cell genome or established as a persistentcytoplasmic element at this early stage of development, it wouldbe possible for all cells derived from the infected zygote to be infected. Consequently a congenitally infected chicken could beconsidered a virus-infected cell clone. The aim of this study wasto ascertain the extent of infection in congenitally infected birds.

Representative samples of all types of tissue from congenitallyinfected chickens and embryos were examined, and only thepresence of clearly resolved virus buds was accepted as evidenceof virus infection in any cell. The only type of tissue that consistently failed to yield evidence of virus multiplication wasnervous tissue. Some other cell types, such as the glandularepithelium of the thyroid follicle and reticular cells of the bonemarrow, also failed to yield evidence of virus multiplication; however, similar tissues in other organs did reveal the presence ofvirus buds, e.g., glandular epithelium of pancreas, intestine, andother organs and primitive reticular cells of the spleen showedample evidence of virus budding.

While the presence of a virus bud on a cell membrane constitutes conclusive evidence that the cell is infected, the absence ofbuds does not necessarily indicate freedom from infection. Thenumber of buds associated with the cells of a given tissue may betoo low for detection in thin sections, and it is possible that someinfected cells may produce little or no virus. The fact that cells

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can be infected and yet reveal no evidence of buds was clearlydemonstrated in skin. Virus multiplication was seen in the epithelial cells of the stratum germinativum and in the deeper cells ofthe stratum spinosum, but no virus buds were found in the moresuperficial cells of the stratum spinosum or in the stratum gran-ulosum. The superficial layers of the skin are formed simply bymigration of cells from the deep layers; therefore, the same cellsthat synthesized virus in detectible quantities when they weredeep in the epidermis failed to do so when they reached the superficial layers. Similarly, other cells that failed to yield evidence ofvirus multiplication, such as cells of nervous tissue, epithelialcells of thyroid, and reticular cells of bone marrow, might nevertheless be infected. Therefore, we can neither confirm nor refutethe hypothesis that all cells of a congenitally infected chickeneither produce virus or contain provirus. It was clear, however,that virus multiplication was far more widespread than had previously been re|>orted.

In congenitally infected chickens, cells derived from all 3primitive germ layers were found to sup])ort ALV multiplication.Growth of virus in cells of ectodermal origin had not been reported previously with any strain of ALV; however, variousrelated avian sarcoma viruses multiplied in the ectodermal layerof the chorioallantoic membrane (14), and Rons sarcoma virusmultiplied in tissue cultures of ectodermal cells from chicken irisepithelium (10). In this study ALV was shown to multiply instratified squamous epithelium of epidermis which is ectodermalin origin. Avian leukosis viruses exhibit oncogenic activity in cellsof mesodermal origin, and various strains of viras have beenshown to reproduce by budding from cell membranes of leukemicerythroblasts (1), myeloblasts (4), and lymphoblasts (8), andfrom other tumor cells of mesodermal origin (13). Further,various strains of ALV have been shown to multiply in non-neoplastic cells of mesodermal origin (8, 13). It has been evidentfor some time that nonneoplastic cells of endodermal origin alsosupport growth of ALV. Some of the earliest and best electronmicrographs of the budding process in ALV infection showedvirus multiplication in pancreatic acinar cells (23), and BAIstrain A (myeloblastosis) virus was shown to reproduce bybudding from hepatic parenchymal cells (5). The striking factbrought out in this study is that many highly differentiated cellsof diverse origin, such as squamous epithelium, muscle, glandularepithelium, etc., are capable of synthesizing ALV.

Only chickens and embryos that appeared healthy were selected for this study, and no evidence of disease, malignant orotherwise, was detected when the animals were dissected. Furthermore, no evidence of cytopathology or cytologie alterations(other than virus budding) was observed in any of the cells thatsupported virus multiplication. Biggs and Payne (2) re[x>rtedthat the F42 strain of ALV, which was used in this study, inducedlymphoid leukosis, erythroleukosis, and nephroblastoma in theirstrains of chickens. In our exi>eriments we used a different strainof chickens, and we observed only lymphoid leukosis in 31 birdsthat died with tumors in the 1st and 2nd congenitally infectedgenerations. Thus, of all the diverse cell types that were infectedwith ALV, only 1 cell type, the lymphoblast, ever showed evidence of malignant transformation. Assuming that erythroleukosis or nephroblastoma eventually would occur in our birds,no more than 3 cell types would be susceptible to ALV-inducedneoplasia. Therefore, ALV appears to have no discernible harmful effect in the vast majority of infected cells. Even the virus-

infected lymphocytes, some of which may be destined to becomeneoplastic in some chickens, were normal in appearance, asidefrom the presence of buds.

These results emphasize the fact that virus infection alone isnot sufficient to account for the induction of neoplasia by ALVand that at least 2 other factors must be taken into account. The1st requirement is that the viras infect an appropriate targetcell, in this case the lymphoblast or perhaps a lymphoblast precursor. It is evident that this occurs at a very early stage ofembryonic development in congenitally infected chickens, buttumors seldom appear before the birds reach 16 weeks of age,and many congenitally infected individuals never develo]) leukosis at all. Some other factor or factors either within the infected cell or in its environment must determine finally whetherand when an infected cell acquires the property of malignancy.In this case development of disease involves a multiple-stepprocess in which (a) the target cell is infected and remains so fora long jjeriod of time without showing signs of malignancy, and(6) the infected cell becomes malignant, resulting in appearanceof the tumor. This can be contrasted with the mechanisms involved with the related Rous sarcoma viras, where initiation ofthe tumor is a single-step event immediately following infection.Elucidation of the events involved in the 2nd phase of ALV infection is clearly one of the central problems of tumor virology.

Acknowledgments

The authors wish to acknowledge the technical assistance ofMrs. U. Feller, who contributed several of the electron micrographs used in this report, and of Mrs. J. Basford and Miss L.Hammock.

References

1. Benedetti, E. L., and Bernhardt, W. Recherches Ultrastruc-turales sur le Virus de la Leucémie Érythroblastique duPoulet, J. Ultrastruct. Res., /: 309-3(5, 1958.

2. Biggs, P. M., and Payne, L. N. Relationship of Marek's Dis

ease (Neural Lymphomatosis) to Lymphoid Leukosis. Nati.Cancer Inst. Monograph, 17: 83-98, 1964.

3. Burmester, B. R. Routes of Natural Infection in Avian Lymphomatosis. Ann. N. Y. Acad. Sci., 68: 487-95, 1957.

4. De Thé,G. Localization and Origin of the Adenosinetriphos-phatase Activity of Avian Myeloblastosis Virus. A Review.Nati. Cancer Inst. Monograph, 17: 651-71, 1964.

5. De Thé,G., Ishiguro, H., Beard, D., and Beard, J. W. Multiplicity of Cell Response to the BAI Strain A (Myeloblastosis)Avian Tumor Virus. VII. Elaboration of Virus by Non-neo-plastic Hepatic Cells. J. Nati. Cancer Inst., 31: 717-29, 191)3.

6. Di Stefano, H. S., and Dougherty, R. M. Virus Multiplicationin the Oviduct of Hens Infected with an Avian Leukosis Virus.Virology, 26: 150-59, 1965.

7. . Mechanisms for Congenital Transmission of AvianLeukosis Virus. J. Nati. Cancer Inst., 37: 869-83, 1966.

8. Dmochowski, L., Grey, C. E., Padgett, F., Langford, P. L.,and Burmester, B. R. Submicroscopic Morphology of AvianNeoplasms. VI. Comparative Studies on Rous Sarcoma, Visceral Lymphomatosis, Erythroblastosis, Myeloblastosis, andNephroblastoma. Texas Kept. Biol. Med., 22: 20-60, 1964.

9. Dougherty, R. M., Simons, P. J., and Chesterman, F. C. Biological Properties of Three Variants of Rous Sarcoma Virus.J. Nati. Cancer Inst., 31: 1285-1307, 1963.

10. Ephrussi, B., and Temin, H. M. Infection of Chick Iris Epithelium with Rous Sarcoma Virus in Vitro. Virology, //: 547-

52, 1960.

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11. Erlandson, R. A. A New Maraglas, D. E. R. 732 Embedment 18. Sarma, P. S. Turner H. C., and Huebner, R. J. An Avian Leu-for Electron Microscopy. J. Cell Biol., Õ2:704-9, 1964. cosis Group-specific Complement Fixation Reaction. Applica-

12. Freeman, J. A., and Spurlock, B. O. A New Epoxy Embedment tion for the Detection and Assay of Non-cytopathogenicfor Electron Microscopy. Ibid., IS: 437-443, 1962. Leucosis Viruses. Virology, 23: 313-21, 1964.

13. Heine, U., De Thé,G., Ishigiiro, H., Sommer, J. H., Beard, D., 19. Sever, J. L. Application of a Microtechnique to Viral Serolog-and Beard, J. W. Multiplicity of Cell Response to the BAI ¡calInvestigations. J. Immunol., 88: 320-29, 1962.Strain A (Myeloblastosis) Avian Tumor Virus. II. Nephro- 20. Spurlock, B. O., Kattine, V. C., and Freeman, J. A. Technicalblastoma (Wilms Tumor): infrastructure. J. Nati. Cancer Modifications in Maraglas Embedding. J. Cell Biol., 17: 203-7Inst., 23: 199-225, 1959. 196314. Keogh, E. V. Ectoderma! Lesions Produced by the Virus of ' R and Ighizaki Reci ocal Pattern of Genetic

Rons Sarcoma. Brit. J. Exptl. Pathol., 19: 1-9, 1938. , . „ ... . „ T. .. . -B, ,, . T-, , ,,. •,,., Resistance to Avian Tumor \ mises in Two Lines of Chickens.15. Luft, J. H. Improvements 111Epoxy Resin Embedding Meth-

D- u T>- u r< »i n Am n ion Virology, 26: 664-72, 1965.ods. J. Biophys. Biochem. Cytol., 9: 409-14, 1961. BJ16 Reynolds, E. S. The Use of Lead Citrate at High pH as an 22' Watson, M. L. Staining of Tissue Sections for Electron Mi-

Electron-opaque Stain in Electron Microscopy. J. Cell Biol., croscopy with Heavy Metals. J. Biophys. Biochem. Cytol.,17: 208-212, 1963. •*•'475-78, 1958.

17. Rubin, H., Cornelius, A., and Fanshier, L. The Pattern of 23. Zeigel, R. F. Morphological Evidence for the Association ofCongenital Transmission of an Avian Leukosis Virus. Proc. Virus Particles with the Pancreatic Acinar Cells of the Chick.Nati. Acad. Sei. U. S., 47: 1058-60, 1961. J. Nati. Cancer Inst., 26: 1011-39,1961.

FIG. 1. Micrograph showing a virus particle (arrow) budding from the sarcolemma of a skeletal muscle fiber (external oblique muscle) in a 14-day chicken congenitally infected with avian leukosis virus (ALV). A region of sarcoplasm (S) and part of a myofibril (M)showing characteristic cross-striations and myofilaments are seen. X 90,700.

FIG. 2. Cross section of portions of 2 cardiac muscle fibers from the left ventricle of the same chicken used in Fig. 1. A virus particle(arrow) is seen budding into space between the 2 fibers. Myofibrils (M) are seen in slightly oblique section. X 90,700.

FIG. 3. Section of smooth muscle from the duodenal muscularis mucosae of an adult chicken congenitally infected with ALV. Portions of 3 cells (S) are seen. A virus particle (arrow) is seen budding at the surface of 1 cell. X 77,100.

FIG. 4. Micrograph demonstrating virus (arrow) budding from the surface of a chondroblast (C) in the developing femur of a congenitally ALV-infected 6-day chick embryo. Free particles are seen in the matrix surrounding the cell. X 74,100.

FIG. 5. Virus particles (arrows) budding from 2 fibroblasts (F) within the pancreas of a congenitally ALV-infected 20-day chicken.X 90,700.

FIG. 6. A virus bud (arrow) developing at the surface of an epidermal cell in the Malpighian layer of skin from an adult hen congenitally infected with ALV. The plasma membranes (pm) of 2 adjacent cells can be traced below the region of the bud but not above,due to a tangential plane of sectioning through the membranes. A portion of the nucleus (;V) of 1 cell and characteristic bundles oftonofilaments (t) are seen. X 90,700.

FIG. 7. Virus particle (arrow) budding from a cytoplasmic extension at the base of a duodenal lining epithelial cell from the samechicken used in Fig. 3. X 84,300.

FIG. 8. Longitudinal section through an interlobular bile duct in the liver of an adult hen congenitally infected with ALV. Portionsof several epithelial cells are seen bordering a lumen. A virus bud (arrow) is seen at the tip of the microvillus that extends into thelumen. X 31,200. Insert represents higher magnification of bud. X 122,500.

FIGS. 9-17. Virus particles (arrows) budding from plasma membranes of various epithelial cells from the same chicken used inFig. 3.

FIG. 9. A virus particle at the surface of a liver parenchymal cell (Li) budding into the space between 2 adjacent cells. X 98,000.FIG. 10. A virus particle budding from a duodenal gland cell (G). X 84,300.FIG. 11. A virus particle budding into a vesicle in the cytoplasm (Cy) of a submandibular salivary gland cell. The mucoserous ma

terial (ms) to be secreted by this cell is seen at the top. X 95,400.FIG. 12. A virus particle budding from the lateral surface of a kidney proximal convoluted tubule cell (PT). Part of an adjacent cell

is seen at the bottom. X 77,500.FIG. 13. A virus particle budding into the lumen (Lu) from a kidney distal convoluted tubule cell (DT). Two other particles are seen

at the left. These are probably virus buds whose points of attachment are in a section above or below the one shown. X 86,100.FIG. 14. Two virus particles budding from a collecting tubule cell (CT) of the kidney into the lumen (Lu). X 77,500.FIG. 15. A virus particle budding into Bowman's space (BS) from a capsular cell (C) of a kidney glomerulus. X 77,500.FIG. 16. A virus particle budding into Bowman's space (BS) from a podocyte (P) of a kidney glomerulus. Note foot processes (/)

extending from this cell to the basement membrane (KM) surrounding a glomerular capillary. Part of an endothelial cell (E) liningthe capillary and the capillary lumen (Lu) are seen. X 77,500.

FIG. 17. A virus particle budding from a cytoreticular cell (Cr) in the bursa of Fabricius. Part of another similar cell is seen above.X 89,500.

FIG. 18. A virus particle (arrow) budding at the plasma membrane of a lymphocyte (L) in the bursa of Fabricius from the sameorgan used in Fig. 17. A portion of the nucleus (N) is seen at lower right. X 89,500.

FIG. 19. A virus particle (arrow) budding from an endothelial cell (E) into the lumen (Lu) of a blood vessel in the cecum of thesame animal used in Fig. 3. X 63,100.

FIG. 20. A virus particle (arrow) budding from a mesothelial cell (Me) of the oviducal visceral peritoneum into the body cavity(BC) of an adult hen congenitally infected with ALV. X 95,400.

FIG. 21. A virus particle (arrow) is seen budding at the surface of a primitive reticular cell (PR) in the spleen of the same animalused in Fig. 3. X 89,500.

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1967;27:322-332. Cancer Res   Robert M. Dougherty and Henry S. Di Stefano  Infected ChickensSites of Avian Leukosis Virus Multiplication in Congenitally

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