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Localization of Herpesvirus of Turkeys in Blood Cells
Localization of Herpesvirus of Turkeys in Blood Cells VIRGINIA L. HINSHAW' and EMILIO C. MORA Department of Poultry Science, Alabama Agricultural Experiment Station, Auburn University, Auburn, Alabama 36830 (Received for publication October 18, 1978)
1980 Poultry Science 59:258-263 INTRODUCTION
1 9 7 2 ; Witter and S o l o m a n , 1 9 7 1 , 1 9 7 2 ) . H V T appeared t o be universally distributed and highly contagious in t u r k e y s , whereas it was n o t in chickens. Witter et al. ( 1 9 7 2 ) d e t e r m i n e d t h a t H V T in t h e peripheral b l o o d of t h e t u r k e y was associated with t h e buffy coat cells and t h a t titers of blood samples from these t u r k e y s ranged from 0 t o 5 9 8 plaque forming u n i t s (PFU) per milliliter ( m e d i a n o f 6 5 P F U / m l ) . T h e incidence of MD has been substantially reduced b y inoculation of y o u n g chicks with H V T obtained from infected chick o r d u c k e m b r y o fibroblasts. Thus, H V T has t h e u n i q u e position of being t h e first virus effectively used commercially as a c o n t r o l against certain t y p e s of cancer (Hilleman, 1972). This s t u d y was t o d e t e r m i n e t h e loci of infection of H V T in t h e circulating b l o o d of its natural host, t h e t u r k e y .
The causative agent of Marek's disease (MD) was f o u n d t o be a herpesvirus, which was highly cell-associated in all tissues of t h e h o s t e x c e p t t h e feather-follicle epithelium (Biggs, 1 9 6 7 ; Biggs and Payne, 1 9 6 7 ; Biggs et al, 1 9 6 8 ; Calnek et al, 1 9 7 0 a , b , 1 9 7 2 ; Purchase, 1 9 7 0 ; Witter et al, 1 9 6 9 ) . T h e intensive s t u d y of MD led t o t h e discovery of a n o t h e r herpesvirus isolated b y Witter et al. ( 1 9 7 0 ) from a p p a r e n t l y h e a l t h y t u r k e y s . T h e t u r k e y isolates p r o d u c e d in chick e m b r y o fibroblasts a syncytial c y t o p a t h o l o g y and t y p e A intranuclear inclusion bodies; plaque f o r m a t i o n was inhibited b y 5-bromo-deoxyuridine. T h e plaques were readily distinguished from those of Marek's disease herpesvirus ( M D H V ) b y their larger size, m o r e rapid appearance, and lytic centers. This agent was classified as a herpesvirus of t u r k e y s (HVT) and was a p p a r e n t l y identical t o a t u r k e y virus isolated earlier b y K a w a m u r a et al. ( 1 9 6 9 ) .
MATERIALS AND METHODS
Witter et al. ( 1 9 7 2 ) r e p o r t e d isolation in tissue culture of cell-associated H V T from t h e buffy coat, k i d n e y , bursa, spleen, and t h y m u s of HVT-infected t u r k e y s and cell-free H V T from quill tips and skin. This g r o u p postulated t h a t H V T replicated in t h e feather follicle epithelium as did M D H V . H V T was considered n o n p a t h o g e n i c for t u r k e y s and chickens (Paul et al, 1 9 7 2 ; Witter,
1 Present address: St. Jude Children's Research Hospital, Memphis, TN 38101.
Ten Broad Breasted Large White t u r k e y s over 1 year in age were selected on t h e basis of t h e H V T t i t e r in t h e b l o o d as d e t e r m i n e d b y plaque counts o n chick e m b r y o fibroblast m o n o l a y e r s . Twelve milliliters of b l o o d were o b t a i n e d from t h e brachial wing vein of each bird. T e n milliliters were m i x e d in tubes with 100 USP units of heparin and 2 ml of blood were allowed t o clot. W i n t r o b e h e m a t o c r i t t u b e s were filled with t h e heparinized blood and centrifuged at 3,500 g for 10 min. The plasma was removed a n d t h e buffy coat from each t u b e was recovered and pooled for each bird. The buffy coat pools for each bird were recentrifuged
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ABSTRACT The buffy coat pellets of blood clots from Broad Breasted Large White turkeys were processed for electron microscopy. No viruses were found in extracellular spaces nor in the heterophils, eosinophils, or basophils. Enveloped virions, measuring approximately 135 nm in diameter consisting of a core 30 to 35 nm in diameter and a capsid 20 to 30 nm thick were found in cytoplasmic vacuoles in lymphocytes. No developmental stages of virus maturation were observed in the nuclei or cytoplasm of any of the lymphocytes. Virus-like particles were observed in the nuclei and perinuclear zones of many erythrocytes and were approximately 135 nm in diameter but did not possess herpesvirus morphology.
HVT IN BLOOD CELLS
in conical tubes for 10 min at 4,500 g until firm pellets were formed which were suspended in 3% glutaraldehyde for 12 hr at 4 C, rinsed in .2 m phosphate buffer for 12 hr at 4 C, then post-fixed in 1 % osmium tetroxide at 4 C for 2 hr according to Millonig (1961). The specimens were minced to 1 mm 3 then dehydrated through graded ethanol and propylene oxide and embedded in plastic capsules in an Araldite 502-Epon 812 mixture according to Mollenhauer (1963). Sections for microscopy were stained according to Reynolds (1963). Small specimens of clotted blood were processed the same way for electron microscopy.
Sections of blood cell specimens from each turkey were examined for the presence of intracellular and/or extracellular viruses. The sections of the clot were made specifically to increase the likelihood of observing any extracellular viruses; however, no extracellular viruses were observed in any of the specimens during this investigation. The most frequently observed leukocyte was the mature heterophil. The heterophils possessed distinctive lobulated nuclei and cytoplasmic primary and secondary granules. The heterophils appeared completely normal in every respect and no viruses were observed in these cells. Several eosinophils were seen in all of the specimens. Most of them possessed very round, homogeneous granules without electron dense centers which was indicative of young, but completely normal, eosinophils. No viruses were observed in these cells. The very few basophils observed did not contain detectable viruses. The lymphocytes from all turkeys characteristically had the same morphology; however, cytoplasmic vacuoles usually contained virus-like particles. A morphology characteristic of herpesviruses was observed in the virus-like particles at high magnification. The virus diameter ranged from 125 to 135 nm and consisted of a core measuring 30 to 35 nm in diameter, a capsid 20 to 30 nm thick, and an envelope 20 to 25 nm thick. All of the viruses were mature, enveloped, and within the vacuole (Fig. 1 , 2 , 3 , 4 ) . The paramyelin figures in these cells were in close proximity to the vacuole; however, no actual connection or association between the two was observed (Fig. 5, 6). All cells in which viruses were observed were identified as lymphocytes. The cells in which
the nuclei were not visible and viruses were present were classified as non-granular leukocytes, probably of the lymphocytic series. Large, granular structures were osberved adjacent to the virus-filled vacuoles in several of the lymphocytes. Some lymphocytes contained mature viruses with the morphology of herpesviruses in a cytoplasmic tubule which curved toward the cell surface; however, no connection of the tubule to the cell membrane was observed in different sections of this cell. Erythrocytes were examined and virus-like particles were found in the nuclei and perinculear regions of some erythrocytes. The virus-like particles appeared to be entering or exiting the nuclei in several places. One virus-like particle was seen in the cytoplasm. Analysis of the particles revealed a morphology unlike that of the viruses in the lymphocytes. The virus-like particles in erythrocytes were 136 nm in diameter and consisted of an electron dense core of 30 nm in diameter, an electron-translucent layer 14 nm thick, an electron-dense layer 16 nm thick, and an envelope-like layer 24 nm thick (Fig. 7, 8,9).
DISCUSSION Evaluation of the data obtained from this electron microscopic study led to several interesting and novel conclusions as to the existence of HVT in its natural host, the turkey. Of the granulocytes, the phagocytic heterophils were considered the most likely to contain viruses. The absence of viruses in the heterophils, as well as in eosinophils and basophils, indicated that the circulating granulocytes were not involved in this particular viral infection or at least not at this stage. The monocytes did not contain viruses; however, a few did contain multimembrane aggregations of paramyelin figures. Paramyelin figures have been reported as common byproducts in many viral infections (Dalton and Haguenau, 1962; Lunger and Maddux, 1972). However, Dowben (1971) stated that these figures were formed from debris, mostly lipid in nature, as a result of phagocytosis of bacteria or other objects by leukocytes. The extreme vacuolization of several monocytes was considered normal for aged cells. Evaluation of the presence of paramyelin figures in the phagocytic monocytes and the absence of
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RESULTS
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FIG. 1. Cytoplasmic vacuole in lymphocyte containing virions with a diameter of 130 to 140 nm having a distinct core (C), capsid (Ca) and envelope (E). X 103,740. FIG. 2. The cytoplasm of a lymphocyte with two virions (arrows) enclosed in separate membrane-bound vacuoles and two electron dense granular bodies. X 66,690. FIG. 3. Three virions adjacent to the nucleus (N) with the core, capsid, and envelope of the virions clearly visible. X 103,740. FIG. 4. A lymphocyte with a myelin figure (M) and two vacuoles, one with six distinct virions and the other with indistinct virus-like particles (arrows). X 13,338.
viruses suggested that monocytes were not the primary source of HVT in the blood. Examination of the turkeys with high HVT
titers revealed many lymphocytes with paramyelin figures and virus-like particles in the cytoplasm. The frequently observed paramyelin
HVT IN BLOOD CELLS
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Downloaded from http://ps.oxfordjournals.org/ at NERL on May 16, 2015 FIG. 5. Lymphocyte or monocyte with myelin figure (M) in nuclear concavity. X 26,676. FIG. 6. A cell, probably a lymphocyte, with a myelin figure (M) adjacent to a large vacuole filled with virions (arrow) and a lymphocyte in the upper portion of the micrograph with one virus in a cytoplasmic vacuole. X26.676. FIG. 7. The cytoplasm of an erythrocyte with several vacuoles and the nucleus with several virus-like particles (arrows). X 20,748. FIG. 8. Two virus-like particles (arrow) in an erythrocyte nucleus. Particle diameter approximately 135 — 140 nm. X 103,740. FIG. 9. A virus-like particle (arrow) in the cytoplasm of an erythrocyte. A clear area surrounding the particle extends from the perinuclear space to near the cell surface. X 51,870.
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HINSHAW AND MORA and mitochondria (Plummer, 1967). Similar vacuoles were observed in MDHV-infected tissue culture cells in which large and prominent membrane-bound cytoplasmic inclusions containing almost exclusively enveloped virus particles were characterized as virus traps to reduce the spread of infection (Ahmed and Schidlovsky, 1968; Nazerian and Burmester, 1968). The segregation of viruses within tubules and vesicles of endoplasmic reticulum and then secretion of hydrolytic enzymes into these areas could reflect a protective mechanism on the part of the cell (Plummer, 1967); this description correlated well with the lymphocytic vacuoles in this study, especially with the presence of lysome-like bodies and myelin figures adjacent to the vacuoles. These HVT-infected lymphocytes, although slightly smaller in diameter, possessed a striking resemblance to the virocytes seen in many human diseases (Litwins and Leibowitz, 1951). The presence of virus-like particles in the erythrocytes represented an unexpected discovery. These particles were consistently located in the nucleus or perinuclear zone of these cells, except for one observed in the cytoplasm. The morphology of these particles was unlike that for HVT in the lymphocytes even though the diameters of the two were approximately the same. The difficulty in obtaining definitive micrographs of these particles frustrated attempts to conclusively classify them as viruses. Also, the possibility that these particles could be cross-sections of fibrils extending from the nuclei to the cell suface of erythrocytes had to be considered; however, it seemed unlikely that fibrils would possess the morphology demonstrated in several of the micrographs.
REFERENCES Ahmed, M., and G. Schidlovsky, 1968. Electron microscopic localization of herpesvirus-type particles in Marek's disease. J. Virol. 2:1443. Biggs, P. M., 1967. Marek's disease. Vet. Record 81:583-592. Biggs, P. M., A. E. Churchill, D. G. Rootes, and R. C. Chubb, 1968. The etiology of Marek's disease- an oncogenic herpes-type virus. Perspectives in Virol. 6:211-237. Biggs, P. M., and L. N. Payne, 1967. Studies on Marek's disease. I. Experimental transmission. J. Nat. Cancer Inst. 39:267-280. Calnek, B. W., H. K. Adldinger, and D. E. Kahn, 1970a. Feather follicle epithelium, a source of enveloped and infectious cell-free herpesvirus from Marek's disease. Avian Dis. 14:219—233.
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figures in lymphocytes which have not been officially classed as phagocytic cells might have been of viral significance. Unusual cytoplasmic vacuoles filled with viruses were observed in numerous lymphocytes. The viruses in the lymphocytes were always enclosed in vacuoles in the cytoplasm and possessed a consistent morphology- an enveloped virus approximately 135 nm thick and an envelope 20 to 25 nm thick. The diameter of these viruses was slightly smaller than the diameter of 150 to 180 given by Nazerian et al. (1971) for HVT in tissue culture with the main variation being in envelope measurements. The nuclei of the infected lymphocytes were examined closely for changes most frequently related to herpesvirus replication in tissue culture, such as margination of the nuclear chromatin, the presence of viral nucleocapsids, and the presence of small nuclear particles 35 nm in diameter (Nazerian et al, 1971). Stages of viral assembly, as nucleocapsids or small nuclear particles, were not seen in any of the nuclei. Even when the number of viruses in the cytoplasm was quite high, the nuclei demonstrated no outstanding abnormalities. HVT in the infected lymphocytes were always present as enveloped virions enclosed in membranebound vacuoles which varied in shape and size. No intermediate stages of virus development were observed in any of the cells. This finding indicated that the viruses were present probably as a result of some type of specialized phagocytosis; however, the presence of so many viruses in the lymphocytes and the complete absence in the most actively phagocytic leukocytes provided little support for this idea. It is possible that phagocytic leukocytes break down the ingested virions. Herpesviruses supposedly exit the nuclei of infected cells through vacuoles formed from indentation of the nuclear membrane continuous with the perinuclear cisternae or through the cisternae of the endoplasmic reticulum (Jawetz et al, 1970; Nii et al, 1968). Examination of micrographs of the lymphocytes suggested that the viruses were within the endoplasmic reticulum. Details of the intracellular development of human cytomegalovirus, another herpesvirus, in tissue culture revealed that this virus left the nucleus in out-pouchings of the endoplasmic reticulum and tended to accumulate in the Golgi particles along with an electron dense material, thus forming the cytoplasmic inclusion body which was frequently surrounded by lysosomes
HVT IN BLOOD CELLS
mester, 1971. Ultrastructural studies of a herpesvirus of turkeys antigenically related to Marek's disease virus. Virology 43:442—452. Nii, S., C. Morgan, and H. M. Rose, 1968. Electron microscopy of herpes simplex virus. J. Virol. May:517-536. Paul. P., C. T. Larsen, M. C. Kumar, and B. S. Pomeroy, 1972. Preliminary observations on egg transmission of turkey herpesvirus (HVT) in turkeys. Avian Dis. 1 6 : 2 7 - 3 3 . Plummer, G., 1967. Comparative virology of the herpes group. Progr. Med. Virol. 9:302—340. Purchase, H. G., 1970. Virus-specific immunofluorescent and precipitin antigens and cell-free virus in tissues of birds infected with Marek's disease. Cancer Res. 30:1898-1908. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electorn opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Witter, R. L., 1972. Turkey herpesvirus: lack of oncogenicity for turkeys. Avian Dis. 16:666— 670. Witter, R. L., G. H. Burgoyne, and J. J. Soloman, 1969. Evidence for herpesvirus as an etiologic agent of Marek's disease. Avian Dis. 13:171—184. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of cell-associated herpesvirus antigenically related to Marek's disease virus. Amer. J. Vet. Res. 31: 525-538. Witter, R. L., K. Nazerian, and J . J . Soloman, 1972. Studies on the in vivo replication of turkey herpesvirus. J. Nat. Cancer Inst. 49:1121—1130. Witter, R. L., and J . J . Soloman, 1971. Epidemiology of a herpesvirus of turkeys (HVT): Possible sources and spread of infection in turkey flocks. Infect, and Immun. 4:356—361. Witter, R. L., and J. J. Soloman, 1972. Experimental infection of turkeys and chickens with herpesvirus of turkeys (HVT). Avian Dis. 16:34—44.
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Calnek, B. W., C. Garrido, W. Okazaki, and I. V. Patrascu, 1972. In vitro methods of assay of turkey herpesvirus. Avian Dis. 16:52—56. Calnek, B. W., T. Ubertini, and H. K. Adldinger, 1970b. Viral antigen, virus particles, and infectivity of tissues from chickens with Marek's disease. J. Nat. Cancer Inst. 45:341—352. Dalton, A. J., and F. Haguenau, ed., 1962. Tumors induced by viruses: Ultrastructural studies. Academic Press, New York. Dowben, Robert M., 1971. Cell biology. Harper & Row, New York. Hilleman, M. R., 1972. Marek's disease vaccine: its implication in biology and medicine. Avian Dis. 16:191-197. Jawetz, E., J. L. Melnick, and E. A. Adelberg, 1970. Review of medical microbiology. Lange Med. Publ., Los Altos, CA. Kawamura, H., D. J. King, Jr., and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13:853— 863. Litwins, J., and S. Leibowitz, 1961. Abnormal lymphocytes "virocytes" in virus disease other than infectious mononucleosis. Acta Haemat. 5: 223-231. Lunger, P. D., and T. C. Maddux, 1972. Fine structure of the avian infectious bursal agent. 1. In vivo viral morphogenesis. J. Avian Dis. 16:874—893. Millonig, G., 1961. Advantages of a phospate buffer for O s 2 0 4 solution in fixation. J. Appl. Phys. 32:1637. Mollenhauer, H. H., 1963. Plastic embedding mixture for use in electron microscopy. J. Stain Tech. 39:111-114. Nazerian, K., and B. R. Burmester, 1968. Electron microscopy of a herpesvirus associated with the agent of Marek's disease in a cell culture. Cancer Res. 28:2454-2462. Nazerian, K., L. F. Lee, R. L. Witter, and B. R. Bur-
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1980 Poultry Science 59:258-263 INTRODUCTION
1 9 7 2 ; Witter and S o l o m a n , 1 9 7 1 , 1 9 7 2 ) . H V T appeared t o be universally distributed and highly contagious in t u r k e y s , whereas it was n o t in chickens. Witter et al. ( 1 9 7 2 ) d e t e r m i n e d t h a t H V T in t h e peripheral b l o o d of t h e t u r k e y was associated with t h e buffy coat cells and t h a t titers of blood samples from these t u r k e y s ranged from 0 t o 5 9 8 plaque forming u n i t s (PFU) per milliliter ( m e d i a n o f 6 5 P F U / m l ) . T h e incidence of MD has been substantially reduced b y inoculation of y o u n g chicks with H V T obtained from infected chick o r d u c k e m b r y o fibroblasts. Thus, H V T has t h e u n i q u e position of being t h e first virus effectively used commercially as a c o n t r o l against certain t y p e s of cancer (Hilleman, 1972). This s t u d y was t o d e t e r m i n e t h e loci of infection of H V T in t h e circulating b l o o d of its natural host, t h e t u r k e y .
The causative agent of Marek's disease (MD) was f o u n d t o be a herpesvirus, which was highly cell-associated in all tissues of t h e h o s t e x c e p t t h e feather-follicle epithelium (Biggs, 1 9 6 7 ; Biggs and Payne, 1 9 6 7 ; Biggs et al, 1 9 6 8 ; Calnek et al, 1 9 7 0 a , b , 1 9 7 2 ; Purchase, 1 9 7 0 ; Witter et al, 1 9 6 9 ) . T h e intensive s t u d y of MD led t o t h e discovery of a n o t h e r herpesvirus isolated b y Witter et al. ( 1 9 7 0 ) from a p p a r e n t l y h e a l t h y t u r k e y s . T h e t u r k e y isolates p r o d u c e d in chick e m b r y o fibroblasts a syncytial c y t o p a t h o l o g y and t y p e A intranuclear inclusion bodies; plaque f o r m a t i o n was inhibited b y 5-bromo-deoxyuridine. T h e plaques were readily distinguished from those of Marek's disease herpesvirus ( M D H V ) b y their larger size, m o r e rapid appearance, and lytic centers. This agent was classified as a herpesvirus of t u r k e y s (HVT) and was a p p a r e n t l y identical t o a t u r k e y virus isolated earlier b y K a w a m u r a et al. ( 1 9 6 9 ) .
MATERIALS AND METHODS
Witter et al. ( 1 9 7 2 ) r e p o r t e d isolation in tissue culture of cell-associated H V T from t h e buffy coat, k i d n e y , bursa, spleen, and t h y m u s of HVT-infected t u r k e y s and cell-free H V T from quill tips and skin. This g r o u p postulated t h a t H V T replicated in t h e feather follicle epithelium as did M D H V . H V T was considered n o n p a t h o g e n i c for t u r k e y s and chickens (Paul et al, 1 9 7 2 ; Witter,
1 Present address: St. Jude Children's Research Hospital, Memphis, TN 38101.
Ten Broad Breasted Large White t u r k e y s over 1 year in age were selected on t h e basis of t h e H V T t i t e r in t h e b l o o d as d e t e r m i n e d b y plaque counts o n chick e m b r y o fibroblast m o n o l a y e r s . Twelve milliliters of b l o o d were o b t a i n e d from t h e brachial wing vein of each bird. T e n milliliters were m i x e d in tubes with 100 USP units of heparin and 2 ml of blood were allowed t o clot. W i n t r o b e h e m a t o c r i t t u b e s were filled with t h e heparinized blood and centrifuged at 3,500 g for 10 min. The plasma was removed a n d t h e buffy coat from each t u b e was recovered and pooled for each bird. The buffy coat pools for each bird were recentrifuged
258
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ABSTRACT The buffy coat pellets of blood clots from Broad Breasted Large White turkeys were processed for electron microscopy. No viruses were found in extracellular spaces nor in the heterophils, eosinophils, or basophils. Enveloped virions, measuring approximately 135 nm in diameter consisting of a core 30 to 35 nm in diameter and a capsid 20 to 30 nm thick were found in cytoplasmic vacuoles in lymphocytes. No developmental stages of virus maturation were observed in the nuclei or cytoplasm of any of the lymphocytes. Virus-like particles were observed in the nuclei and perinuclear zones of many erythrocytes and were approximately 135 nm in diameter but did not possess herpesvirus morphology.
HVT IN BLOOD CELLS
in conical tubes for 10 min at 4,500 g until firm pellets were formed which were suspended in 3% glutaraldehyde for 12 hr at 4 C, rinsed in .2 m phosphate buffer for 12 hr at 4 C, then post-fixed in 1 % osmium tetroxide at 4 C for 2 hr according to Millonig (1961). The specimens were minced to 1 mm 3 then dehydrated through graded ethanol and propylene oxide and embedded in plastic capsules in an Araldite 502-Epon 812 mixture according to Mollenhauer (1963). Sections for microscopy were stained according to Reynolds (1963). Small specimens of clotted blood were processed the same way for electron microscopy.
Sections of blood cell specimens from each turkey were examined for the presence of intracellular and/or extracellular viruses. The sections of the clot were made specifically to increase the likelihood of observing any extracellular viruses; however, no extracellular viruses were observed in any of the specimens during this investigation. The most frequently observed leukocyte was the mature heterophil. The heterophils possessed distinctive lobulated nuclei and cytoplasmic primary and secondary granules. The heterophils appeared completely normal in every respect and no viruses were observed in these cells. Several eosinophils were seen in all of the specimens. Most of them possessed very round, homogeneous granules without electron dense centers which was indicative of young, but completely normal, eosinophils. No viruses were observed in these cells. The very few basophils observed did not contain detectable viruses. The lymphocytes from all turkeys characteristically had the same morphology; however, cytoplasmic vacuoles usually contained virus-like particles. A morphology characteristic of herpesviruses was observed in the virus-like particles at high magnification. The virus diameter ranged from 125 to 135 nm and consisted of a core measuring 30 to 35 nm in diameter, a capsid 20 to 30 nm thick, and an envelope 20 to 25 nm thick. All of the viruses were mature, enveloped, and within the vacuole (Fig. 1 , 2 , 3 , 4 ) . The paramyelin figures in these cells were in close proximity to the vacuole; however, no actual connection or association between the two was observed (Fig. 5, 6). All cells in which viruses were observed were identified as lymphocytes. The cells in which
the nuclei were not visible and viruses were present were classified as non-granular leukocytes, probably of the lymphocytic series. Large, granular structures were osberved adjacent to the virus-filled vacuoles in several of the lymphocytes. Some lymphocytes contained mature viruses with the morphology of herpesviruses in a cytoplasmic tubule which curved toward the cell surface; however, no connection of the tubule to the cell membrane was observed in different sections of this cell. Erythrocytes were examined and virus-like particles were found in the nuclei and perinculear regions of some erythrocytes. The virus-like particles appeared to be entering or exiting the nuclei in several places. One virus-like particle was seen in the cytoplasm. Analysis of the particles revealed a morphology unlike that of the viruses in the lymphocytes. The virus-like particles in erythrocytes were 136 nm in diameter and consisted of an electron dense core of 30 nm in diameter, an electron-translucent layer 14 nm thick, an electron-dense layer 16 nm thick, and an envelope-like layer 24 nm thick (Fig. 7, 8,9).
DISCUSSION Evaluation of the data obtained from this electron microscopic study led to several interesting and novel conclusions as to the existence of HVT in its natural host, the turkey. Of the granulocytes, the phagocytic heterophils were considered the most likely to contain viruses. The absence of viruses in the heterophils, as well as in eosinophils and basophils, indicated that the circulating granulocytes were not involved in this particular viral infection or at least not at this stage. The monocytes did not contain viruses; however, a few did contain multimembrane aggregations of paramyelin figures. Paramyelin figures have been reported as common byproducts in many viral infections (Dalton and Haguenau, 1962; Lunger and Maddux, 1972). However, Dowben (1971) stated that these figures were formed from debris, mostly lipid in nature, as a result of phagocytosis of bacteria or other objects by leukocytes. The extreme vacuolization of several monocytes was considered normal for aged cells. Evaluation of the presence of paramyelin figures in the phagocytic monocytes and the absence of
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RESULTS
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FIG. 1. Cytoplasmic vacuole in lymphocyte containing virions with a diameter of 130 to 140 nm having a distinct core (C), capsid (Ca) and envelope (E). X 103,740. FIG. 2. The cytoplasm of a lymphocyte with two virions (arrows) enclosed in separate membrane-bound vacuoles and two electron dense granular bodies. X 66,690. FIG. 3. Three virions adjacent to the nucleus (N) with the core, capsid, and envelope of the virions clearly visible. X 103,740. FIG. 4. A lymphocyte with a myelin figure (M) and two vacuoles, one with six distinct virions and the other with indistinct virus-like particles (arrows). X 13,338.
viruses suggested that monocytes were not the primary source of HVT in the blood. Examination of the turkeys with high HVT
titers revealed many lymphocytes with paramyelin figures and virus-like particles in the cytoplasm. The frequently observed paramyelin
HVT IN BLOOD CELLS
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Downloaded from http://ps.oxfordjournals.org/ at NERL on May 16, 2015 FIG. 5. Lymphocyte or monocyte with myelin figure (M) in nuclear concavity. X 26,676. FIG. 6. A cell, probably a lymphocyte, with a myelin figure (M) adjacent to a large vacuole filled with virions (arrow) and a lymphocyte in the upper portion of the micrograph with one virus in a cytoplasmic vacuole. X26.676. FIG. 7. The cytoplasm of an erythrocyte with several vacuoles and the nucleus with several virus-like particles (arrows). X 20,748. FIG. 8. Two virus-like particles (arrow) in an erythrocyte nucleus. Particle diameter approximately 135 — 140 nm. X 103,740. FIG. 9. A virus-like particle (arrow) in the cytoplasm of an erythrocyte. A clear area surrounding the particle extends from the perinuclear space to near the cell surface. X 51,870.
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HINSHAW AND MORA and mitochondria (Plummer, 1967). Similar vacuoles were observed in MDHV-infected tissue culture cells in which large and prominent membrane-bound cytoplasmic inclusions containing almost exclusively enveloped virus particles were characterized as virus traps to reduce the spread of infection (Ahmed and Schidlovsky, 1968; Nazerian and Burmester, 1968). The segregation of viruses within tubules and vesicles of endoplasmic reticulum and then secretion of hydrolytic enzymes into these areas could reflect a protective mechanism on the part of the cell (Plummer, 1967); this description correlated well with the lymphocytic vacuoles in this study, especially with the presence of lysome-like bodies and myelin figures adjacent to the vacuoles. These HVT-infected lymphocytes, although slightly smaller in diameter, possessed a striking resemblance to the virocytes seen in many human diseases (Litwins and Leibowitz, 1951). The presence of virus-like particles in the erythrocytes represented an unexpected discovery. These particles were consistently located in the nucleus or perinuclear zone of these cells, except for one observed in the cytoplasm. The morphology of these particles was unlike that for HVT in the lymphocytes even though the diameters of the two were approximately the same. The difficulty in obtaining definitive micrographs of these particles frustrated attempts to conclusively classify them as viruses. Also, the possibility that these particles could be cross-sections of fibrils extending from the nuclei to the cell suface of erythrocytes had to be considered; however, it seemed unlikely that fibrils would possess the morphology demonstrated in several of the micrographs.
REFERENCES Ahmed, M., and G. Schidlovsky, 1968. Electron microscopic localization of herpesvirus-type particles in Marek's disease. J. Virol. 2:1443. Biggs, P. M., 1967. Marek's disease. Vet. Record 81:583-592. Biggs, P. M., A. E. Churchill, D. G. Rootes, and R. C. Chubb, 1968. The etiology of Marek's disease- an oncogenic herpes-type virus. Perspectives in Virol. 6:211-237. Biggs, P. M., and L. N. Payne, 1967. Studies on Marek's disease. I. Experimental transmission. J. Nat. Cancer Inst. 39:267-280. Calnek, B. W., H. K. Adldinger, and D. E. Kahn, 1970a. Feather follicle epithelium, a source of enveloped and infectious cell-free herpesvirus from Marek's disease. Avian Dis. 14:219—233.
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figures in lymphocytes which have not been officially classed as phagocytic cells might have been of viral significance. Unusual cytoplasmic vacuoles filled with viruses were observed in numerous lymphocytes. The viruses in the lymphocytes were always enclosed in vacuoles in the cytoplasm and possessed a consistent morphology- an enveloped virus approximately 135 nm thick and an envelope 20 to 25 nm thick. The diameter of these viruses was slightly smaller than the diameter of 150 to 180 given by Nazerian et al. (1971) for HVT in tissue culture with the main variation being in envelope measurements. The nuclei of the infected lymphocytes were examined closely for changes most frequently related to herpesvirus replication in tissue culture, such as margination of the nuclear chromatin, the presence of viral nucleocapsids, and the presence of small nuclear particles 35 nm in diameter (Nazerian et al, 1971). Stages of viral assembly, as nucleocapsids or small nuclear particles, were not seen in any of the nuclei. Even when the number of viruses in the cytoplasm was quite high, the nuclei demonstrated no outstanding abnormalities. HVT in the infected lymphocytes were always present as enveloped virions enclosed in membranebound vacuoles which varied in shape and size. No intermediate stages of virus development were observed in any of the cells. This finding indicated that the viruses were present probably as a result of some type of specialized phagocytosis; however, the presence of so many viruses in the lymphocytes and the complete absence in the most actively phagocytic leukocytes provided little support for this idea. It is possible that phagocytic leukocytes break down the ingested virions. Herpesviruses supposedly exit the nuclei of infected cells through vacuoles formed from indentation of the nuclear membrane continuous with the perinuclear cisternae or through the cisternae of the endoplasmic reticulum (Jawetz et al, 1970; Nii et al, 1968). Examination of micrographs of the lymphocytes suggested that the viruses were within the endoplasmic reticulum. Details of the intracellular development of human cytomegalovirus, another herpesvirus, in tissue culture revealed that this virus left the nucleus in out-pouchings of the endoplasmic reticulum and tended to accumulate in the Golgi particles along with an electron dense material, thus forming the cytoplasmic inclusion body which was frequently surrounded by lysosomes
HVT IN BLOOD CELLS
mester, 1971. Ultrastructural studies of a herpesvirus of turkeys antigenically related to Marek's disease virus. Virology 43:442—452. Nii, S., C. Morgan, and H. M. Rose, 1968. Electron microscopy of herpes simplex virus. J. Virol. May:517-536. Paul. P., C. T. Larsen, M. C. Kumar, and B. S. Pomeroy, 1972. Preliminary observations on egg transmission of turkey herpesvirus (HVT) in turkeys. Avian Dis. 1 6 : 2 7 - 3 3 . Plummer, G., 1967. Comparative virology of the herpes group. Progr. Med. Virol. 9:302—340. Purchase, H. G., 1970. Virus-specific immunofluorescent and precipitin antigens and cell-free virus in tissues of birds infected with Marek's disease. Cancer Res. 30:1898-1908. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electorn opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Witter, R. L., 1972. Turkey herpesvirus: lack of oncogenicity for turkeys. Avian Dis. 16:666— 670. Witter, R. L., G. H. Burgoyne, and J. J. Soloman, 1969. Evidence for herpesvirus as an etiologic agent of Marek's disease. Avian Dis. 13:171—184. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of cell-associated herpesvirus antigenically related to Marek's disease virus. Amer. J. Vet. Res. 31: 525-538. Witter, R. L., K. Nazerian, and J . J . Soloman, 1972. Studies on the in vivo replication of turkey herpesvirus. J. Nat. Cancer Inst. 49:1121—1130. Witter, R. L., and J . J . Soloman, 1971. Epidemiology of a herpesvirus of turkeys (HVT): Possible sources and spread of infection in turkey flocks. Infect, and Immun. 4:356—361. Witter, R. L., and J. J. Soloman, 1972. Experimental infection of turkeys and chickens with herpesvirus of turkeys (HVT). Avian Dis. 16:34—44.
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Calnek, B. W., C. Garrido, W. Okazaki, and I. V. Patrascu, 1972. In vitro methods of assay of turkey herpesvirus. Avian Dis. 16:52—56. Calnek, B. W., T. Ubertini, and H. K. Adldinger, 1970b. Viral antigen, virus particles, and infectivity of tissues from chickens with Marek's disease. J. Nat. Cancer Inst. 45:341—352. Dalton, A. J., and F. Haguenau, ed., 1962. Tumors induced by viruses: Ultrastructural studies. Academic Press, New York. Dowben, Robert M., 1971. Cell biology. Harper & Row, New York. Hilleman, M. R., 1972. Marek's disease vaccine: its implication in biology and medicine. Avian Dis. 16:191-197. Jawetz, E., J. L. Melnick, and E. A. Adelberg, 1970. Review of medical microbiology. Lange Med. Publ., Los Altos, CA. Kawamura, H., D. J. King, Jr., and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13:853— 863. Litwins, J., and S. Leibowitz, 1961. Abnormal lymphocytes "virocytes" in virus disease other than infectious mononucleosis. Acta Haemat. 5: 223-231. Lunger, P. D., and T. C. Maddux, 1972. Fine structure of the avian infectious bursal agent. 1. In vivo viral morphogenesis. J. Avian Dis. 16:874—893. Millonig, G., 1961. Advantages of a phospate buffer for O s 2 0 4 solution in fixation. J. Appl. Phys. 32:1637. Mollenhauer, H. H., 1963. Plastic embedding mixture for use in electron microscopy. J. Stain Tech. 39:111-114. Nazerian, K., and B. R. Burmester, 1968. Electron microscopy of a herpesvirus associated with the agent of Marek's disease in a cell culture. Cancer Res. 28:2454-2462. Nazerian, K., L. F. Lee, R. L. Witter, and B. R. Bur-
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