Morphogenesis of Marek's Disease Virus in Feather Follicle Epithelium
1,2
E. A. Johnson,3,4 C. N. Burke,3 T. N. Fredrickson," and R. A. DiCapua 5 SUMMARY-Three evaluative systems, immunodiffusion, fluorescent antibody (FA), and electron microscopy (EM), were used to follow the morphogenesis of Marek's disease virus in inoculated chickens. Of the three, EM and FA were the most sensitive in detecting early stages of infection. Virus particles were found in skin biopsy specimens as early as 12 days post inoculation. Immature naked particles appeared first in the nucleus; later particles were enveloped in the cytoplasm and enclosed in cytoplasmic inclusion bodies. No evidence for continued virus replication was seen in feather follicles after an initial burst of heavy virus production, which lasted several weeks. Residual virus, however, was found occasionally in cy· toplasmic inclusion bodies within keratinized material near the feathers. This was believed to contribute to the long-term shedding of infectious virus into the environment.-J Natl Cancer Inst 55: 89-99, 1975.
SURVIVAL OF MAREK'S DISEASE VIRUS (MDV) in susceptible flocks of domestic fowl depends on a number of factors, three of which are: 1) protective enclosure of the virus in cytoplasmic inclusion bodies (1, 2) , 2) continued shedding of virus-infected feather follicle epithelial (FFE) cells from surviving carrier birds (3, 4), and 3) maintenance of infectivity of the virus long after such cells have been shed into the environment (5-7). Various methods have been used in attempts to detect the virus in specific tissues, to identify its maturation sites, and to determine factors contributing to its longterm survival in the environment. Fluorescent antibody (FA) and immunodiffusion (ID) tests have been used by Calnek and Hitchner (8) to demonstrate soluble antigen in FFE. Using chick kidney cells, Nazerian and Witter (1) found that infectious virus was recoverable from cell-free extracts of feather follicles and, by light microscopy, noted specific cytopathic alterations in the tissue. In addition, Calnek and associates (2) clearly observed enveloped particles after electron microscope (EM) examination of negatively stained extracts of follicle epithelium. The extended survival of the virus, whether shed in follicle epithelium (5) or carried in particulate airborne matter (5-7), has been well documented. Contact transmission from apparently healthy recovered birds to susceptible ones has been reported (3, 4), with successful transmissions occurring as long as a year and more following recovery. The survival of this virus parallels that reported for other viruses having cutaneous involvement (rabbit papilloma, herpes zoster, and smallpox). EXPERIMENTAL DESIGN
Because a detailed study of MDV maturation in individual birds has not yet been reported, attempts were made to follow the morphogenesis with repeated skin biopsies of individual birds over a period of time. To study the correlation and sensitivity of several methods of virus detection, EM examinations as well as FA and ID tests were done on tissues from the same biopsy. In some instances, biopsies were continued until estimates of the decline in virus production could be made.
Experiment 1.-In this experiment, 10 birds were inoculated as described under "Materials and Methods." ID tests were done on biopsy samples removed from each bird on days 12 through 17 post inoculation. Biopsy tissues from 3 birds were selected for further study and examined by EM and FA (table 1). Two survivors from this experiment were examined by ID 93 days after exposure for soluble antigens and by EM to provide materials for study from recovered birds. Experiment 2.-This study was designed to estimate the level of skin antigen in individual birds for several weeks. For this group, 18 birds were inoculated as described under "Materials and Methods." Weekly biopsy tissues were taken from the second through the ninth week and tested by ID for skin antigen. Since the tests on tissues from surviving birds gave negative results (no precipitin bands), EM examinations were performed on the biopsy tissues held in reserve to ascertain the level of virus production (table 1). MATERIALS AND METHODS
Virus.-A cell-free preparation of the €onn-B strain of MDV (9) was obtained by extraction of virus from skins of chickens 4 weeks after inoculation (10). Infected birds were skinned and the feathers and subcutaneous fat removed. Skins were homogenized in phosphate-buffered saline (PBS) and sonicated at 95-100 W for 3 minutes to disrupt the cells and release the virus. The material was filtered through sterile gauze and centrifuged at I,OOOxg for 20 minutes to remove whole cells. The supernatant was spun at 10,000xg for 20-30 minutes, and the 10,000xg supernatant was used as inoculum. Experimental birds.-Day-old White Leghorn chickens from a specific pathogen-free flock at the University of Connecticut (11) were inoculated intratracheally with 0.1 ml skin extract. Chickens were housed in positive-pressure isolators described by Prince et al. (12). Birds were anesthesized with ether before biopsy samples were taken. A fold of skin along the ventral feather tract was drawn together with sutures and clipped away. Each biopsy specimen containing 6-12 follicles was divided, and representative numbers of feather follicles were processed for EM, homogenized, extracted for soluble antigen for ID tests, and then frozen for direct staining with FA. Electron mieroscopy.-Skin biopsy tissues were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at 4° C. The tissue was washed twice with 0.1 M phosphate buffer (pH 7.3), postfixed in 2% osmium tetroxide, dehydrated in increasing concentrations of ethyl alcohol and propylene oxide, and embedded in Epon-Araldite Received November 15, 1974; accepted March 14, 1975. Scientific Contribution No. 598, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Conn. 06268. 3 Department of Pathobiology, University of Connecticut. Address reprint requests to Dr. C. N. Burke. 4 Present address: Department of Biology, Wesleyan University, Middletown, Conn. 06457. 5 College of Pharmacy, University of Connecticut. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
89
90
JOHNSON, BURKE, FREDRICKSON, AND DlCAPUA TABLE
I.-Examination schedule/or experiments 1 and f!! Tests perNumber formed of on birds biopsy tested material
Experiment
Number of birds/ group
Biopsy schedule
1
10
Daily biopsies, days 1217; again on day 92
EMB ID
Weekly biopsies, weeks 3-9
ID EMc
2
18
FAb
3/10 2/10 2/10 18/18 4/18
• Morphogenesis was followed in 3/10 birds (#17394, 17395, 17396). Selection for EM followed ID testing for Marek's disease (MD) skin antigen. Birds 17394 and 17395 were ID positive for MD antigen, whereas 17396 developed no detectable antigen during the testing period. • FA staining was limited to birds #17394 and 17395. , Feather fol1icles from 4 birds which had shown an increase and subsequent decrease in ID antigen were thin sectioned and examined for MDV.
(13). Thick sections of embedded FFE were cut and prepared with toluidine blue and Paragon multiple stain; areas were marked for thin sectioning on the basis of lesion development. Thin sections were cut on the LKB Ultratome and picked up on 100-mesh grids coated with 0.25% Formvar. They were double stained with uranyl acetate and lead citrate (14) and examined with the .J eo1co T6S electron microscope. ID assay.-Skin containing feather follicles was homogenized in Ten Broeck grinders in PBS (pH 6.8) to give a 10% weight/volume suspension. The homogenate was frozen and thawed four times and tested for virusspecific antigen. The skin extract used as the inoculum for these birds also served as a positive antigen control. Serum from SPAFAS, Inc. (Norwich, Conn.), obtained from chickens which had been naturally infected with MD, was used as the positive antiserum. The antiserum was placed in the central well and the antigens in the eight outer wells. Slides were placed in a moist chamber at 27° C and the results recorded at 24 and 48 hours. FA tests.-Frozen sections 6 iJ. thick, including cross sections of several follicles, were cut in a cryostat. Sections were air dried, fixed in acetone for 10 minutes, and covered with undiluted fluorescein-conjugated serum from convalescent MD-infected chickens supplied by Dr. B. Calnek (Cornell University, Ithaca, N.Y.). Sections were incubated for 30 minutes at 37° C, washed in three changes of PBS (pH 7.2) and once in bicarbonate buffer (pH 9.5) , mounted in 10% glycerine in bicarbonate buffer, and immediately examined with the fluorescence microscope; the intensity of fluorescence was rated from 0 to + 3. RESULTS Viral Morphogenesis in FFE
Changes in follicular epithelium after MDV infection did not progress sequentially from small lesions on day 12 to large, deeply involved lesions on day 17 post inoculation. Rather, sections of several follicles on each day contained lesions at various stages of development, which probably indicated that the entire epithelium was not infected simultaneously but during a span of several days. The following is a composite picture from 6 days' observation of 3 birds, [from the 12th to 18th day post inoculation with MDV (expt. 1)]. Virus particles first appeared in the nucleus of keratinizing cells of the stratum transivativum (1) (fig. 1).
Their appearance was followed by the develo{Jment of a nuclear inclusion characterized by margination of the chromatin and development of a less dense homogeneous material in the center of the nucleus. Virions were occasionally grouped in clusters of 4 or 5 but were usually scattered singly throughout the inclusion (fig. 2A) . An envelope around the immature nuclear particles was acquired at three sites within the infected cell. Enveloped virus particles were infrequently seen between the inner and outer lamellae of the nuclear membrane (fig. 2B). Within the nucleus itself, an occasional group of enveloped virions were near the nuclear membrane (fig. 2C). These particles did not represent the type of mature virions normally produced in such quantities in FFE cells. Enveloped nuclear particles were 150-180 nm in diameter, and the envelopes enclosed no extraviral material between capsid and envelope. They resembled the particles characterized by Nazerian (1!5) that are produced in MDV-infected cell culture. Envelopment occurred primarily in the cytoplasm of epithelial cells. Immature virions, released through typically enlarged nuclear pores (fig. 2B) or breaks in the nuclear membrane into the cytoplasm, were embedded in clumps of an electron-dense substance. Unit membranes formed de novo around this material enclosing the virus nucleoid and a portion of the electron-dense material within the developing envelope (figs. 2A, 2D) to produce a virion measuring 210-280 nm in diameter. The cytoplasmic content of infected cells varied considerably, perhaps as a function of the duration of infection in each cell. Inclusion body material appeared in the cytoplasm after the production of nuclear virus was underway and followed closely on the envelopment of the immature virus in the cytoplasm. Inclusion body material was more granular and/ generally less dense than the cytoplasmic material enclosed by the virus envelope. The material appeared as small granules that accumulated in the cytoplasm and coalesced; one or several already mature viruses were enclosed in the process (fig. 3A). An increase in the volume of inclusion body material in the cell was followed by degeneration of the cytoplasm and lysis of the cell membrane (fig. 3B) . The nucleus remained intact throughout virus production, release, and cytoplasmic encapsulation but degenerated after rupture of the cell membrane, with the subsequent release of immature virus particles into the mass of cell debris from surrounding cells (fig. 4) . Whether some of these released particles then formed envelopes from material present in the cell debris was not clear. During the first stages of virus multiplication, virus particles were in the outer two layers of epithelium in the stratum transivativum and stratum corneum (fig. 5). In the keratinized cells of the stratum corneum (containing naked and less frequently mature virions), the virus could complete replication, and the cells continued to differentiate into more of the same type. As the lesion progressed, however, cells of the intermediary and germinal layers became infected and cell differentiation no longer took place. A recognizable germinal layer was no longer evident and the four layers of epithelium were replaced by acellular material composed of mature virus, inclusion bodies, and cell debris (fig. 6). Regeneration of the follicular epithelium followed the period of virus production. In many instances, more frequently toward the end of the biopsy period, the lesion
91
SURVIVAL OF MAREK'S DISEASE VIRUS
was separated from the regenerating follicle by uninfected differentiating cells (fig. 7) and forced toward the feather shaft by new layers of uninfected stratum corneum (fig. 8) . Correlation of FA, 10, and EM Tests
Cross sections of follicles from the 2 most heavily infected birds, # 17394 and 17395, indicated progressive spread of fluorescent antigen over the surface of follicles from the 12th to the 15th day post inoculation (table 2). From EM observation, bird # 17394 showed a more limited degree of follicular involvement than bird # 17395, and this was reflected in reaction of tissue with FA. By comparison, positive ID results were not obtained until day 15, when large quantities of inclusion body material and mature virus were observed in the epithelium. Distribution of fluorescent antigen over the layers of epithelium varied. In many areas, fluorescence spread from the basal layers to keratinized cells at the follicle surface; in others, only the upper areas of the follicle, the stratum transivativum, and stratum corneum fluoresced; in a few regions, single cells or small groups of cells within the intermediary layers showed fluorescence. TABLE 2.-Experiment 1: presence of MDV in FFE as measured by ID, EM, and FA tests
Bird No.
Test used
1738717393
lD
17394
lD EM
FA
17395 17396
Days post inoculation 12
13
14
15
16
17
0/7
0/7
0/7
2/7
2/7
2/7
-
-
-
+
+
+ 0
+2
-
+1
+ NT" NT
-
-
+ +2
-
FA
+ +2
+ +3
+ + +3
+ + NT
+ + NT
lD EM
-
NT
+
NT
+
+
lD EM
-
+ +1
52
• NT - not tested,
Distribution of infected cells was similar in thin sections from adjacent follicles. Termination of Shedding: Experiment 2
Biopsy material from birds # 17394, 17395, and 17396, examined for virus on days 12-17 in experiment I, was obtained again 92 days post inoculation. Birds # 17394 and 17395, previously positive for virus particles and soluble skin antigen, were negative for both by day 92 and had developed MDV serum antibodies. These samples suggested that virus production had dropped to a low level or terminated in birds which at one time were heavy virus shedders. To determine the length of time that virus was actively produced in large quantities by cells of the follicular epithelium, biopsies were performed weekly until death of the bird or disappearance of soluble antigen in skin extracts (table 3) . Of the 18 birds tested, 17 developed skin antigen, and II of these died during the experiment. Skin antigen and serum antibodies were present in all II at the last biopsy, and postmortem examination revealed heart, kidney, and spleen tumors typically found. Soluble antigen could be detected in the skin of surviving birds for periods ranging from 4 to 7 weeks. After this time, skin antigen either disappeared or became undetectable with the ID test, and serum antibodies developed. To determine the extent of lesion reduction occurring with the loss of skin antigen, EM examinations were made of follicles taken at the last positive ID test and the following week when soluble antigen could no longer be detected by ID (table 4). Four birds were examined, as indicated in table 3. Skin antigen-positive samples from all 4 birds contained mature MDV particles. In II of 12 follicles, MDV particles were limited to the highly keratinized layers of the stratum corneum. Particles were frequent in only I keratinized cell, but some follicles contained large deposits of inclusion bodies and cell debris at the surface. These infected areas were always separated from the underlying epithelial layers by a layer of keratinized cells. In only I follicle of the 12 examined was the lesion still associated with the stratum transivativum. Macrophage infiltration between
TABLE 3.-Experiment 2: persistence of skin antigen as determined by ID testing of individual birds Bird No. 19659 19661 19663 19664 19693 19656 19657 19658 19660 19665 19667 19668 19669 19671 19675 19677 19684 19685
Days post inoculation a 53
62
+
+
+D
(- )
+
D
+
D
12
13
14
15
17
21
24
31
38
-
+
+ +
+ -
+ + + +
+ D + +
+
+
+
+ + +
+
+ +
+ + +
+ + + +
(+ )
(- )
+ (+ )
(- )
+
+
+ D + + D +
D
-
+ + + + +
D -
(+ )
+ +
+ +
-
+
+ +
-
-
+ +
-
-
-
-
-
+ + + + + + +
-
-
-
(+)
-
(- )
47 D
D
-
+ D +
• D - died during testing period; all birds had developed visceral tumors characteristic of MD. Plus and minus signs in parenth•••• indicate biopsy samples examined for virus particles by EM.
92
JOHNSON, BURKE, FREDRICKSON, AND D1CAPUA TABLE
4.-MD virus particles in FFE as related to ID tests ID-positive biopsy
Bird No.
ID-negative biopsy
Week Virus in Virus in Week Virus in Virus in post germinal keratin- post germinal keratininoculayer ized inoculayer ized layer lation layer lation
19656 19667 19669 19684
5 6 6 5
0/3 1/3 0/3 0/3
3/3 3/3 3/3 1/3
6 7 7 6
0/3 0/3 0/3 0/3
0/3 1/3 1/3 0/3
cells of the germinal layer was extensive in most follicles. All these observations indicated progressive terminal lesions. Biopsies taken the following week from these same birds, by then ID negative, were devoid of any sign of virus multiplication in the germinal layers. In 2 birds (#19656, 19684) all layers were free of immature and mature virus particles, while I follicle from each of 2 other birds (#19667, 19669) contained I small inclusion body and associated enveloped virus in the mass of keratinized material at its surface (fig. 9) . Here, also, infiltration of macrophages between cells of the germinal and intermediate layer was common. DISCUSSION
Comparison of ID, FA, and EM tests of MD-infected feather follicles showed FA and EM to be the most sensitive methods for detection of the virus. Positive results from FA-stained follicles correlated well with adjacent follicles from the same bird examined by EM. Positive ID results, however, were obtained only when EM revealed extensive lesion development. Virus replication of MDV within the nucleus was typical of the herpesvirus (16). The addition of an envelope for most herpesviruses takes place within the nucleus, at the nuclear membrane, or through a process of budding. This type of envelopment has also been described for MDV in cell culture (15, 17), and Ahmed and Schidlovsky (18) have made similar observations for MDV in cytoplasmic inclusions. In follicular epithelium, however, most viruses were released from the nucleus and acquired an envelope through de novo membrane synthesis in the cytoplasm. The electron-dense material incorporated into the mature virus during membrane formation appeared in the cytoplasm in association with the newly released nuclear virus and preceded the formation of inclusion body material. In older cells, large quantities of both products accumulated in the cytoplasm of infected cells. Purchase (19) has reported fluorescent antigen in follicular epithelium as early as the fifth day post inoculation with MDV. We observed virus reproduction on day 12 and, in 1 bird examined, the virus was replicating over nearly 100% of the surface epithelium by the 15th day post inoculation. The virus did not reproduce in all birds examined to the same extent or as soon after infection as in some. However, the fact that all but I bird (table 3) responded with a postive ID test between the second and third week post inoculation indicated that lesion development had progressed to a sufficiently advanced state to be measured with this relatively insensitive test. EM examination of infected follicles showed that the virus infected several successive generations of
germinal cells and replicated as these cells differentiated; this produced a multilayered lesion containing mature virus in the keratinized cells at the surface and in relatively undifferentiated cells within the lesion. Underlying many of these lesions were areas where the epithelium had regenerated. Cells of the germinal layer were again capable of producing epithelial cells that completed differentiation without virus replication. This regenerative process, with a subsequent reduced level of integumentary virus, took place over a period of several weeks (expt. 2), as indicated by ID and EM examination, and may be related to the rising level of precipitin antibody. Bankowski et al. (20) found that a 2week delay occurred between the appearance of the antigen and the production of antibody. Although this antibody had no effect on visceral lesion formation or regression or on the final percent mortality of birds with MD (4), Ahmed et al. (21) demonstrated the specificity of the precipitin antibody for the virus particle itself. A fibrillar coat formed around both the virus capsid and envelope following reaction of the antibody with the virus. If the precipitin antibody produced after MD infection can inactivate non-cell-associated virus, it may also limit the number of virions capable of reaching the follicular epithelium and initiating virus replication at sites in these layers. Earlier work by Witter et al. (4) indicates that chickens, previously infected with MDV, still contain minimal quantities of integumentary virus at 76 weeks and are capable of contact transmission to other birds. Whether this is the result of loss of residual inclusion body material trapped along the feather shaft or sporadic production of new virus in isolated cells along the follicular epithelium is still unanswered. Our results show a dramatic decrease in the extent of follicular involvement after an initial burst of virus production. This is followed by regeneration of follicular tissue and loss of inclusion bodies containing mature virus. Residual inclusion body material and virions are in the stratum corneum of these birds, and there seems no doubt that this material, carrying infectious virus, is released into the environment and is the main source of the contamination. REFERENCES
(1) NAZERIAN K, WITIER RL: Cell-free transmission and in vivo replication of Marek's disease virus. J Virol 5:38&-397, 1970 (2) CALNEK B\Y, Am.DINGER HK, KAHS DE: Feather follicle epithelium: A source of enveloped and infectious cell-free herpesvirus from Marek's disease. Avian Dis 14:219-233, 1970 (3) KENZY SG, CHO BR: Transmission of classical Marek's disease by affected and carrier birds. Avian Dis 13:211-214, 1969 (4) WITIER RL, SOLOMON H- CHAMPION LR, et al: Long-term studies of Marek's disease infection in individual chickens. Avian Dis 15:346--365, 1971 (5) CARROZZA J: Aerobiological transmission of Marek's disease virus. Ph.D. Thesis, Univ Conn., Storrs, Conn., 1973 (6) WITIER RL, BURGOYNE GH, BURMESTER BR: Survival of Marek's disease agent in litter and droppings. Avian Dis 12:522-530, 1968 (7) BEASLEY IN, PATTERSON LT, MCWADE DH: Transmission of Marek's disease by poultry house dust and chicken dander, Am J Vet Res 31:339-344, 1970 (8) CALNEK BW, HITCHNER SB: Localization of viral agent in chickens infected with Marek's disease herpesvirus. J Nat! Cancer Inst 43:935-949, 1969 (9) JAKOWSKI RM, FREDRICKSON TN, CHOMIAK TW, et al: Hematopoietic destruction in Marek's disease. Avian Dis 14:384385, 1970
SURVIVAL OF MAREK'S DISEASE VIRUS
(10) CARROZZA J, FREDRICKSON TN, PRINCE RP, et al: Transmission of Marek's disease by shedder chickens: Extraction of virus and soluble antigen from skin. Am J Vet Res 33:1499--1506, 1972 (11) LUGINBUHL RE, HOLDENREID R, STEVENSON RE: Establishment and maintenance of a specific pathogen-free (SPF) flock of White Leghorn chickens. Prog Immunobiol Stand 3:6-13, 1969 (12) PRINCE RP, FREDRICKSON TN, CARROZZA J: A disposable isolation chamber for use in avian disease research. Poult Sci 49:1746-1748, 1970 (13) MOLLENHAUER HH: Plastic embedding mixture for use in electron microscopy. Stain Technol 39:111-114, 1964 (14) REYNOLDS ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Bioi 17:208-212, 1963 (15) NAZERIAN K: Further studies on the replication of Marek's disease virus in the chicken and in cell culture. J Natl Cancer Inst 47:207-217, 1971
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(16) KAPLAN AS (ed.): The Herpesviruses. New York, Academic
Press, 1973
(17) NAZERIAN K, BURMESTER BR: Electron microscopy of a herpes(18) (19)
(20)
(21)
virus associated with the agent of Marek's disease in cell culture. Cancer Res 28:2454-2462, 1968 AHMED M, ScHIDLOVSKY G: Electron microscopic localization of herpesvirus-type particles in Marek's disease. J Virol 2:1443-1457, 1968 PURCHASE HG: Virus-specific immunofluorescent and precipitin antigens and cell-free virus in the tissues of birds infected with Marek's disease. Cancer Res 30: 1898-1908, 1970 BANKOWSKI RA, MIKAMI T, REYNOLDS B: The relation between infection of chickens with Marek's disease and the presence of precipitin antibodies. Avian Dis 14:723-737, 1970 AHMED M, JENSEN KE, SLATTERY SM, et al: Detection of Marek's disease herpesvirus antigen by fluorescent and coating antibody. Avian Dis 14:349--363, 1970
I.-Section of feather follicle showing portion of stratum corneum at tipper left; stratum transivativum and intermediary layer are at lower right. Cell-associated virus particles are just below first keratinized cell. Inclusion body formation has not taken place, though mature enveloped particles are within the cell. X 7,500
FIGURE
94
JOHNSON, BURKE, FREDRICKSON, AND DlCAPUA
2.-A) Immature MDV particles in nucleus (upper left) and cytoplasm of infected cell. Virus is associated with dense material in cytoplasm, and a membrane is forming around several particles (arrow). Nuclear particles exhibit a variety of core shapes; cytoplasmic particles contain a more condensed nucleoid. X 36,000. B) Enveloped particles without a nucleoid between inner and outer nuclear membrane. Large nuclear pore is visible (arrow) and a mature particle is in cytoplasm near pore. X 28,750. C) Section through well-developed nuclear inclusion body containing several enveloped virus particles near nuclear membrane. Envelope measures 150-180 nm and contains no extraviral material. These particles were occasional in nuclei but never in cytoplasm or inclusion bodies. X 48,000. D) Immature virus associated with dense material in cytoplasm adjacent to nucleus (upper right) . Section is from bird having the least epithelial involvement at 14 days post inoculation. X 36,000
FIGURE
JOHNSON, B.URKE, FREDRICKSON, AND DICAPUA
95
o
3.-A) Section from a bird with heavy cutaneous involvement, 17 days post inoculation. Nucleus contains immature particles and large pores at upper and lower margins. Cytoplasm consists of clumps of inclusion body material varying in size and density. Only a few mature virions are embedded in larger inclusions. X 19,500. B) Section of cell debris at surface of lesion, 16 days post inoculation. Size of inclusion body has increased, enclosing many mature virions. Nucleus at left is still intact. X 17,000
FIGURE
96
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
5 4.--eell debris from lysed cells at surface of follicle. Material contains many immature virions (arrows) released following nuclear lysis. X 14,000
FIGURE
5.-Several layers of follicular epithelium from bird on 12th day post inoculation, illustrating virus multiplication in successive layers of follicle cells. Virus particles are limited to stratum transivativum. X 8,625
FIGURE
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
97
FIGURE 6.-Follicle from most heavily infected bird at 17 days post inoculation. Intact cell at left is part of basal membrane. Cell layers. including the stratum germanitivum, have been replaced by large inclusion bodies, cell debris, and mature virus. X 12,600 FIGURE 7.-Portions of 2 follicles regenerating new layers of keratinized cells beneath lesion. Nucleus in lower left contains immature nuclear virus, indicating possible reinfection of follicle. X 12,600
98
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
FIGURE 8.-Small inclusion body remaining at follicular surface. Underlying cells gave no indication of renewed infection with MDV. X 12,600 FIGURE 9.-Follicle from a bird in which MDV precipitin antigen had dropped below detectable level. Keratinized cells, at surface of follicle (left) do not contain virus particles. Virus is present in older cells (right), which have moved from surface of follicle toward feather shaft. X 6,355
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
99
1,2
E. A. Johnson,3,4 C. N. Burke,3 T. N. Fredrickson," and R. A. DiCapua 5 SUMMARY-Three evaluative systems, immunodiffusion, fluorescent antibody (FA), and electron microscopy (EM), were used to follow the morphogenesis of Marek's disease virus in inoculated chickens. Of the three, EM and FA were the most sensitive in detecting early stages of infection. Virus particles were found in skin biopsy specimens as early as 12 days post inoculation. Immature naked particles appeared first in the nucleus; later particles were enveloped in the cytoplasm and enclosed in cytoplasmic inclusion bodies. No evidence for continued virus replication was seen in feather follicles after an initial burst of heavy virus production, which lasted several weeks. Residual virus, however, was found occasionally in cy· toplasmic inclusion bodies within keratinized material near the feathers. This was believed to contribute to the long-term shedding of infectious virus into the environment.-J Natl Cancer Inst 55: 89-99, 1975.
SURVIVAL OF MAREK'S DISEASE VIRUS (MDV) in susceptible flocks of domestic fowl depends on a number of factors, three of which are: 1) protective enclosure of the virus in cytoplasmic inclusion bodies (1, 2) , 2) continued shedding of virus-infected feather follicle epithelial (FFE) cells from surviving carrier birds (3, 4), and 3) maintenance of infectivity of the virus long after such cells have been shed into the environment (5-7). Various methods have been used in attempts to detect the virus in specific tissues, to identify its maturation sites, and to determine factors contributing to its longterm survival in the environment. Fluorescent antibody (FA) and immunodiffusion (ID) tests have been used by Calnek and Hitchner (8) to demonstrate soluble antigen in FFE. Using chick kidney cells, Nazerian and Witter (1) found that infectious virus was recoverable from cell-free extracts of feather follicles and, by light microscopy, noted specific cytopathic alterations in the tissue. In addition, Calnek and associates (2) clearly observed enveloped particles after electron microscope (EM) examination of negatively stained extracts of follicle epithelium. The extended survival of the virus, whether shed in follicle epithelium (5) or carried in particulate airborne matter (5-7), has been well documented. Contact transmission from apparently healthy recovered birds to susceptible ones has been reported (3, 4), with successful transmissions occurring as long as a year and more following recovery. The survival of this virus parallels that reported for other viruses having cutaneous involvement (rabbit papilloma, herpes zoster, and smallpox). EXPERIMENTAL DESIGN
Because a detailed study of MDV maturation in individual birds has not yet been reported, attempts were made to follow the morphogenesis with repeated skin biopsies of individual birds over a period of time. To study the correlation and sensitivity of several methods of virus detection, EM examinations as well as FA and ID tests were done on tissues from the same biopsy. In some instances, biopsies were continued until estimates of the decline in virus production could be made.
Experiment 1.-In this experiment, 10 birds were inoculated as described under "Materials and Methods." ID tests were done on biopsy samples removed from each bird on days 12 through 17 post inoculation. Biopsy tissues from 3 birds were selected for further study and examined by EM and FA (table 1). Two survivors from this experiment were examined by ID 93 days after exposure for soluble antigens and by EM to provide materials for study from recovered birds. Experiment 2.-This study was designed to estimate the level of skin antigen in individual birds for several weeks. For this group, 18 birds were inoculated as described under "Materials and Methods." Weekly biopsy tissues were taken from the second through the ninth week and tested by ID for skin antigen. Since the tests on tissues from surviving birds gave negative results (no precipitin bands), EM examinations were performed on the biopsy tissues held in reserve to ascertain the level of virus production (table 1). MATERIALS AND METHODS
Virus.-A cell-free preparation of the €onn-B strain of MDV (9) was obtained by extraction of virus from skins of chickens 4 weeks after inoculation (10). Infected birds were skinned and the feathers and subcutaneous fat removed. Skins were homogenized in phosphate-buffered saline (PBS) and sonicated at 95-100 W for 3 minutes to disrupt the cells and release the virus. The material was filtered through sterile gauze and centrifuged at I,OOOxg for 20 minutes to remove whole cells. The supernatant was spun at 10,000xg for 20-30 minutes, and the 10,000xg supernatant was used as inoculum. Experimental birds.-Day-old White Leghorn chickens from a specific pathogen-free flock at the University of Connecticut (11) were inoculated intratracheally with 0.1 ml skin extract. Chickens were housed in positive-pressure isolators described by Prince et al. (12). Birds were anesthesized with ether before biopsy samples were taken. A fold of skin along the ventral feather tract was drawn together with sutures and clipped away. Each biopsy specimen containing 6-12 follicles was divided, and representative numbers of feather follicles were processed for EM, homogenized, extracted for soluble antigen for ID tests, and then frozen for direct staining with FA. Electron mieroscopy.-Skin biopsy tissues were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at 4° C. The tissue was washed twice with 0.1 M phosphate buffer (pH 7.3), postfixed in 2% osmium tetroxide, dehydrated in increasing concentrations of ethyl alcohol and propylene oxide, and embedded in Epon-Araldite Received November 15, 1974; accepted March 14, 1975. Scientific Contribution No. 598, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, Conn. 06268. 3 Department of Pathobiology, University of Connecticut. Address reprint requests to Dr. C. N. Burke. 4 Present address: Department of Biology, Wesleyan University, Middletown, Conn. 06457. 5 College of Pharmacy, University of Connecticut. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO. I, JULY 1975
89
90
JOHNSON, BURKE, FREDRICKSON, AND DlCAPUA TABLE
I.-Examination schedule/or experiments 1 and f!! Tests perNumber formed of on birds biopsy tested material
Experiment
Number of birds/ group
Biopsy schedule
1
10
Daily biopsies, days 1217; again on day 92
EMB ID
Weekly biopsies, weeks 3-9
ID EMc
2
18
FAb
3/10 2/10 2/10 18/18 4/18
• Morphogenesis was followed in 3/10 birds (#17394, 17395, 17396). Selection for EM followed ID testing for Marek's disease (MD) skin antigen. Birds 17394 and 17395 were ID positive for MD antigen, whereas 17396 developed no detectable antigen during the testing period. • FA staining was limited to birds #17394 and 17395. , Feather fol1icles from 4 birds which had shown an increase and subsequent decrease in ID antigen were thin sectioned and examined for MDV.
(13). Thick sections of embedded FFE were cut and prepared with toluidine blue and Paragon multiple stain; areas were marked for thin sectioning on the basis of lesion development. Thin sections were cut on the LKB Ultratome and picked up on 100-mesh grids coated with 0.25% Formvar. They were double stained with uranyl acetate and lead citrate (14) and examined with the .J eo1co T6S electron microscope. ID assay.-Skin containing feather follicles was homogenized in Ten Broeck grinders in PBS (pH 6.8) to give a 10% weight/volume suspension. The homogenate was frozen and thawed four times and tested for virusspecific antigen. The skin extract used as the inoculum for these birds also served as a positive antigen control. Serum from SPAFAS, Inc. (Norwich, Conn.), obtained from chickens which had been naturally infected with MD, was used as the positive antiserum. The antiserum was placed in the central well and the antigens in the eight outer wells. Slides were placed in a moist chamber at 27° C and the results recorded at 24 and 48 hours. FA tests.-Frozen sections 6 iJ. thick, including cross sections of several follicles, were cut in a cryostat. Sections were air dried, fixed in acetone for 10 minutes, and covered with undiluted fluorescein-conjugated serum from convalescent MD-infected chickens supplied by Dr. B. Calnek (Cornell University, Ithaca, N.Y.). Sections were incubated for 30 minutes at 37° C, washed in three changes of PBS (pH 7.2) and once in bicarbonate buffer (pH 9.5) , mounted in 10% glycerine in bicarbonate buffer, and immediately examined with the fluorescence microscope; the intensity of fluorescence was rated from 0 to + 3. RESULTS Viral Morphogenesis in FFE
Changes in follicular epithelium after MDV infection did not progress sequentially from small lesions on day 12 to large, deeply involved lesions on day 17 post inoculation. Rather, sections of several follicles on each day contained lesions at various stages of development, which probably indicated that the entire epithelium was not infected simultaneously but during a span of several days. The following is a composite picture from 6 days' observation of 3 birds, [from the 12th to 18th day post inoculation with MDV (expt. 1)]. Virus particles first appeared in the nucleus of keratinizing cells of the stratum transivativum (1) (fig. 1).
Their appearance was followed by the develo{Jment of a nuclear inclusion characterized by margination of the chromatin and development of a less dense homogeneous material in the center of the nucleus. Virions were occasionally grouped in clusters of 4 or 5 but were usually scattered singly throughout the inclusion (fig. 2A) . An envelope around the immature nuclear particles was acquired at three sites within the infected cell. Enveloped virus particles were infrequently seen between the inner and outer lamellae of the nuclear membrane (fig. 2B). Within the nucleus itself, an occasional group of enveloped virions were near the nuclear membrane (fig. 2C). These particles did not represent the type of mature virions normally produced in such quantities in FFE cells. Enveloped nuclear particles were 150-180 nm in diameter, and the envelopes enclosed no extraviral material between capsid and envelope. They resembled the particles characterized by Nazerian (1!5) that are produced in MDV-infected cell culture. Envelopment occurred primarily in the cytoplasm of epithelial cells. Immature virions, released through typically enlarged nuclear pores (fig. 2B) or breaks in the nuclear membrane into the cytoplasm, were embedded in clumps of an electron-dense substance. Unit membranes formed de novo around this material enclosing the virus nucleoid and a portion of the electron-dense material within the developing envelope (figs. 2A, 2D) to produce a virion measuring 210-280 nm in diameter. The cytoplasmic content of infected cells varied considerably, perhaps as a function of the duration of infection in each cell. Inclusion body material appeared in the cytoplasm after the production of nuclear virus was underway and followed closely on the envelopment of the immature virus in the cytoplasm. Inclusion body material was more granular and/ generally less dense than the cytoplasmic material enclosed by the virus envelope. The material appeared as small granules that accumulated in the cytoplasm and coalesced; one or several already mature viruses were enclosed in the process (fig. 3A). An increase in the volume of inclusion body material in the cell was followed by degeneration of the cytoplasm and lysis of the cell membrane (fig. 3B) . The nucleus remained intact throughout virus production, release, and cytoplasmic encapsulation but degenerated after rupture of the cell membrane, with the subsequent release of immature virus particles into the mass of cell debris from surrounding cells (fig. 4) . Whether some of these released particles then formed envelopes from material present in the cell debris was not clear. During the first stages of virus multiplication, virus particles were in the outer two layers of epithelium in the stratum transivativum and stratum corneum (fig. 5). In the keratinized cells of the stratum corneum (containing naked and less frequently mature virions), the virus could complete replication, and the cells continued to differentiate into more of the same type. As the lesion progressed, however, cells of the intermediary and germinal layers became infected and cell differentiation no longer took place. A recognizable germinal layer was no longer evident and the four layers of epithelium were replaced by acellular material composed of mature virus, inclusion bodies, and cell debris (fig. 6). Regeneration of the follicular epithelium followed the period of virus production. In many instances, more frequently toward the end of the biopsy period, the lesion
91
SURVIVAL OF MAREK'S DISEASE VIRUS
was separated from the regenerating follicle by uninfected differentiating cells (fig. 7) and forced toward the feather shaft by new layers of uninfected stratum corneum (fig. 8) . Correlation of FA, 10, and EM Tests
Cross sections of follicles from the 2 most heavily infected birds, # 17394 and 17395, indicated progressive spread of fluorescent antigen over the surface of follicles from the 12th to the 15th day post inoculation (table 2). From EM observation, bird # 17394 showed a more limited degree of follicular involvement than bird # 17395, and this was reflected in reaction of tissue with FA. By comparison, positive ID results were not obtained until day 15, when large quantities of inclusion body material and mature virus were observed in the epithelium. Distribution of fluorescent antigen over the layers of epithelium varied. In many areas, fluorescence spread from the basal layers to keratinized cells at the follicle surface; in others, only the upper areas of the follicle, the stratum transivativum, and stratum corneum fluoresced; in a few regions, single cells or small groups of cells within the intermediary layers showed fluorescence. TABLE 2.-Experiment 1: presence of MDV in FFE as measured by ID, EM, and FA tests
Bird No.
Test used
1738717393
lD
17394
lD EM
FA
17395 17396
Days post inoculation 12
13
14
15
16
17
0/7
0/7
0/7
2/7
2/7
2/7
-
-
-
+
+
+ 0
+2
-
+1
+ NT" NT
-
-
+ +2
-
FA
+ +2
+ +3
+ + +3
+ + NT
+ + NT
lD EM
-
NT
+
NT
+
+
lD EM
-
+ +1
52
• NT - not tested,
Distribution of infected cells was similar in thin sections from adjacent follicles. Termination of Shedding: Experiment 2
Biopsy material from birds # 17394, 17395, and 17396, examined for virus on days 12-17 in experiment I, was obtained again 92 days post inoculation. Birds # 17394 and 17395, previously positive for virus particles and soluble skin antigen, were negative for both by day 92 and had developed MDV serum antibodies. These samples suggested that virus production had dropped to a low level or terminated in birds which at one time were heavy virus shedders. To determine the length of time that virus was actively produced in large quantities by cells of the follicular epithelium, biopsies were performed weekly until death of the bird or disappearance of soluble antigen in skin extracts (table 3) . Of the 18 birds tested, 17 developed skin antigen, and II of these died during the experiment. Skin antigen and serum antibodies were present in all II at the last biopsy, and postmortem examination revealed heart, kidney, and spleen tumors typically found. Soluble antigen could be detected in the skin of surviving birds for periods ranging from 4 to 7 weeks. After this time, skin antigen either disappeared or became undetectable with the ID test, and serum antibodies developed. To determine the extent of lesion reduction occurring with the loss of skin antigen, EM examinations were made of follicles taken at the last positive ID test and the following week when soluble antigen could no longer be detected by ID (table 4). Four birds were examined, as indicated in table 3. Skin antigen-positive samples from all 4 birds contained mature MDV particles. In II of 12 follicles, MDV particles were limited to the highly keratinized layers of the stratum corneum. Particles were frequent in only I keratinized cell, but some follicles contained large deposits of inclusion bodies and cell debris at the surface. These infected areas were always separated from the underlying epithelial layers by a layer of keratinized cells. In only I follicle of the 12 examined was the lesion still associated with the stratum transivativum. Macrophage infiltration between
TABLE 3.-Experiment 2: persistence of skin antigen as determined by ID testing of individual birds Bird No. 19659 19661 19663 19664 19693 19656 19657 19658 19660 19665 19667 19668 19669 19671 19675 19677 19684 19685
Days post inoculation a 53
62
+
+
+D
(- )
+
D
+
D
12
13
14
15
17
21
24
31
38
-
+
+ +
+ -
+ + + +
+ D + +
+
+
+
+ + +
+
+ +
+ + +
+ + + +
(+ )
(- )
+ (+ )
(- )
+
+
+ D + + D +
D
-
+ + + + +
D -
(+ )
+ +
+ +
-
+
+ +
-
-
+ +
-
-
-
-
-
+ + + + + + +
-
-
-
(+)
-
(- )
47 D
D
-
+ D +
• D - died during testing period; all birds had developed visceral tumors characteristic of MD. Plus and minus signs in parenth•••• indicate biopsy samples examined for virus particles by EM.
92
JOHNSON, BURKE, FREDRICKSON, AND D1CAPUA TABLE
4.-MD virus particles in FFE as related to ID tests ID-positive biopsy
Bird No.
ID-negative biopsy
Week Virus in Virus in Week Virus in Virus in post germinal keratin- post germinal keratininoculayer ized inoculayer ized layer lation layer lation
19656 19667 19669 19684
5 6 6 5
0/3 1/3 0/3 0/3
3/3 3/3 3/3 1/3
6 7 7 6
0/3 0/3 0/3 0/3
0/3 1/3 1/3 0/3
cells of the germinal layer was extensive in most follicles. All these observations indicated progressive terminal lesions. Biopsies taken the following week from these same birds, by then ID negative, were devoid of any sign of virus multiplication in the germinal layers. In 2 birds (#19656, 19684) all layers were free of immature and mature virus particles, while I follicle from each of 2 other birds (#19667, 19669) contained I small inclusion body and associated enveloped virus in the mass of keratinized material at its surface (fig. 9) . Here, also, infiltration of macrophages between cells of the germinal and intermediate layer was common. DISCUSSION
Comparison of ID, FA, and EM tests of MD-infected feather follicles showed FA and EM to be the most sensitive methods for detection of the virus. Positive results from FA-stained follicles correlated well with adjacent follicles from the same bird examined by EM. Positive ID results, however, were obtained only when EM revealed extensive lesion development. Virus replication of MDV within the nucleus was typical of the herpesvirus (16). The addition of an envelope for most herpesviruses takes place within the nucleus, at the nuclear membrane, or through a process of budding. This type of envelopment has also been described for MDV in cell culture (15, 17), and Ahmed and Schidlovsky (18) have made similar observations for MDV in cytoplasmic inclusions. In follicular epithelium, however, most viruses were released from the nucleus and acquired an envelope through de novo membrane synthesis in the cytoplasm. The electron-dense material incorporated into the mature virus during membrane formation appeared in the cytoplasm in association with the newly released nuclear virus and preceded the formation of inclusion body material. In older cells, large quantities of both products accumulated in the cytoplasm of infected cells. Purchase (19) has reported fluorescent antigen in follicular epithelium as early as the fifth day post inoculation with MDV. We observed virus reproduction on day 12 and, in 1 bird examined, the virus was replicating over nearly 100% of the surface epithelium by the 15th day post inoculation. The virus did not reproduce in all birds examined to the same extent or as soon after infection as in some. However, the fact that all but I bird (table 3) responded with a postive ID test between the second and third week post inoculation indicated that lesion development had progressed to a sufficiently advanced state to be measured with this relatively insensitive test. EM examination of infected follicles showed that the virus infected several successive generations of
germinal cells and replicated as these cells differentiated; this produced a multilayered lesion containing mature virus in the keratinized cells at the surface and in relatively undifferentiated cells within the lesion. Underlying many of these lesions were areas where the epithelium had regenerated. Cells of the germinal layer were again capable of producing epithelial cells that completed differentiation without virus replication. This regenerative process, with a subsequent reduced level of integumentary virus, took place over a period of several weeks (expt. 2), as indicated by ID and EM examination, and may be related to the rising level of precipitin antibody. Bankowski et al. (20) found that a 2week delay occurred between the appearance of the antigen and the production of antibody. Although this antibody had no effect on visceral lesion formation or regression or on the final percent mortality of birds with MD (4), Ahmed et al. (21) demonstrated the specificity of the precipitin antibody for the virus particle itself. A fibrillar coat formed around both the virus capsid and envelope following reaction of the antibody with the virus. If the precipitin antibody produced after MD infection can inactivate non-cell-associated virus, it may also limit the number of virions capable of reaching the follicular epithelium and initiating virus replication at sites in these layers. Earlier work by Witter et al. (4) indicates that chickens, previously infected with MDV, still contain minimal quantities of integumentary virus at 76 weeks and are capable of contact transmission to other birds. Whether this is the result of loss of residual inclusion body material trapped along the feather shaft or sporadic production of new virus in isolated cells along the follicular epithelium is still unanswered. Our results show a dramatic decrease in the extent of follicular involvement after an initial burst of virus production. This is followed by regeneration of follicular tissue and loss of inclusion bodies containing mature virus. Residual inclusion body material and virions are in the stratum corneum of these birds, and there seems no doubt that this material, carrying infectious virus, is released into the environment and is the main source of the contamination. REFERENCES
(1) NAZERIAN K, WITIER RL: Cell-free transmission and in vivo replication of Marek's disease virus. J Virol 5:38&-397, 1970 (2) CALNEK B\Y, Am.DINGER HK, KAHS DE: Feather follicle epithelium: A source of enveloped and infectious cell-free herpesvirus from Marek's disease. Avian Dis 14:219-233, 1970 (3) KENZY SG, CHO BR: Transmission of classical Marek's disease by affected and carrier birds. Avian Dis 13:211-214, 1969 (4) WITIER RL, SOLOMON H- CHAMPION LR, et al: Long-term studies of Marek's disease infection in individual chickens. Avian Dis 15:346--365, 1971 (5) CARROZZA J: Aerobiological transmission of Marek's disease virus. Ph.D. Thesis, Univ Conn., Storrs, Conn., 1973 (6) WITIER RL, BURGOYNE GH, BURMESTER BR: Survival of Marek's disease agent in litter and droppings. Avian Dis 12:522-530, 1968 (7) BEASLEY IN, PATTERSON LT, MCWADE DH: Transmission of Marek's disease by poultry house dust and chicken dander, Am J Vet Res 31:339-344, 1970 (8) CALNEK BW, HITCHNER SB: Localization of viral agent in chickens infected with Marek's disease herpesvirus. J Nat! Cancer Inst 43:935-949, 1969 (9) JAKOWSKI RM, FREDRICKSON TN, CHOMIAK TW, et al: Hematopoietic destruction in Marek's disease. Avian Dis 14:384385, 1970
SURVIVAL OF MAREK'S DISEASE VIRUS
(10) CARROZZA J, FREDRICKSON TN, PRINCE RP, et al: Transmission of Marek's disease by shedder chickens: Extraction of virus and soluble antigen from skin. Am J Vet Res 33:1499--1506, 1972 (11) LUGINBUHL RE, HOLDENREID R, STEVENSON RE: Establishment and maintenance of a specific pathogen-free (SPF) flock of White Leghorn chickens. Prog Immunobiol Stand 3:6-13, 1969 (12) PRINCE RP, FREDRICKSON TN, CARROZZA J: A disposable isolation chamber for use in avian disease research. Poult Sci 49:1746-1748, 1970 (13) MOLLENHAUER HH: Plastic embedding mixture for use in electron microscopy. Stain Technol 39:111-114, 1964 (14) REYNOLDS ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Bioi 17:208-212, 1963 (15) NAZERIAN K: Further studies on the replication of Marek's disease virus in the chicken and in cell culture. J Natl Cancer Inst 47:207-217, 1971
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(16) KAPLAN AS (ed.): The Herpesviruses. New York, Academic
Press, 1973
(17) NAZERIAN K, BURMESTER BR: Electron microscopy of a herpes(18) (19)
(20)
(21)
virus associated with the agent of Marek's disease in cell culture. Cancer Res 28:2454-2462, 1968 AHMED M, ScHIDLOVSKY G: Electron microscopic localization of herpesvirus-type particles in Marek's disease. J Virol 2:1443-1457, 1968 PURCHASE HG: Virus-specific immunofluorescent and precipitin antigens and cell-free virus in the tissues of birds infected with Marek's disease. Cancer Res 30: 1898-1908, 1970 BANKOWSKI RA, MIKAMI T, REYNOLDS B: The relation between infection of chickens with Marek's disease and the presence of precipitin antibodies. Avian Dis 14:723-737, 1970 AHMED M, JENSEN KE, SLATTERY SM, et al: Detection of Marek's disease herpesvirus antigen by fluorescent and coating antibody. Avian Dis 14:349--363, 1970
I.-Section of feather follicle showing portion of stratum corneum at tipper left; stratum transivativum and intermediary layer are at lower right. Cell-associated virus particles are just below first keratinized cell. Inclusion body formation has not taken place, though mature enveloped particles are within the cell. X 7,500
FIGURE
94
JOHNSON, BURKE, FREDRICKSON, AND DlCAPUA
2.-A) Immature MDV particles in nucleus (upper left) and cytoplasm of infected cell. Virus is associated with dense material in cytoplasm, and a membrane is forming around several particles (arrow). Nuclear particles exhibit a variety of core shapes; cytoplasmic particles contain a more condensed nucleoid. X 36,000. B) Enveloped particles without a nucleoid between inner and outer nuclear membrane. Large nuclear pore is visible (arrow) and a mature particle is in cytoplasm near pore. X 28,750. C) Section through well-developed nuclear inclusion body containing several enveloped virus particles near nuclear membrane. Envelope measures 150-180 nm and contains no extraviral material. These particles were occasional in nuclei but never in cytoplasm or inclusion bodies. X 48,000. D) Immature virus associated with dense material in cytoplasm adjacent to nucleus (upper right) . Section is from bird having the least epithelial involvement at 14 days post inoculation. X 36,000
FIGURE
JOHNSON, B.URKE, FREDRICKSON, AND DICAPUA
95
o
3.-A) Section from a bird with heavy cutaneous involvement, 17 days post inoculation. Nucleus contains immature particles and large pores at upper and lower margins. Cytoplasm consists of clumps of inclusion body material varying in size and density. Only a few mature virions are embedded in larger inclusions. X 19,500. B) Section of cell debris at surface of lesion, 16 days post inoculation. Size of inclusion body has increased, enclosing many mature virions. Nucleus at left is still intact. X 17,000
FIGURE
96
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
5 4.--eell debris from lysed cells at surface of follicle. Material contains many immature virions (arrows) released following nuclear lysis. X 14,000
FIGURE
5.-Several layers of follicular epithelium from bird on 12th day post inoculation, illustrating virus multiplication in successive layers of follicle cells. Virus particles are limited to stratum transivativum. X 8,625
FIGURE
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
97
FIGURE 6.-Follicle from most heavily infected bird at 17 days post inoculation. Intact cell at left is part of basal membrane. Cell layers. including the stratum germanitivum, have been replaced by large inclusion bodies, cell debris, and mature virus. X 12,600 FIGURE 7.-Portions of 2 follicles regenerating new layers of keratinized cells beneath lesion. Nucleus in lower left contains immature nuclear virus, indicating possible reinfection of follicle. X 12,600
98
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
FIGURE 8.-Small inclusion body remaining at follicular surface. Underlying cells gave no indication of renewed infection with MDV. X 12,600 FIGURE 9.-Follicle from a bird in which MDV precipitin antigen had dropped below detectable level. Keratinized cells, at surface of follicle (left) do not contain virus particles. Virus is present in older cells (right), which have moved from surface of follicle toward feather shaft. X 6,355
JOHNSON, BURKE, FREDRICKSON, AND DICAPUA
99
