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Distribution of Herpesvirus in Turkeys
ENVIRONMENT AND HEALTH Distribution of Herpesvirus in Turkeys MARGARET BARRETT HEIN and E. C. MORA Auburn University Agricultural Experiment Station, Auburn, Alabama 36830 (Received for publication December 1, 1978)
INTRODUCTION The turkey herpesvirus (HVT) was first isolated from kidney cell cultures of apparently normal turkeys by Kawamura et al. (1969) and the infectious virion was described as being strongly cell associated. Cytopathic effects (CPE) caused by the virion in cell cultures were formation of syncytia, intranuclear inclusions, and small clear plaques. Plaques also were formed on the HVT-inoculated chorio-allantoic membrane of chicken embryos. Inhibition of plaque formation by 5-bromo-2-deoxyuridine suggested the genetic material of the virion to be deoxyribonucleic acid (DNA). Ultrastructural studies revealed that a viral particle contained a dense core measuring 60 nm, a capsid of about 85 x 100 nm, and an envelope 125 X 165 nm in diameter. On the basis of these characteristics, the HVT strain was designated as a Group B herpesvirus (Kawamura etal, 1969; Witter et al, 1970). Witter et al. (1970) isolated a herpesvirus from turkey blood and kidney by inoculating the turkey blood and trypsinized kidney cells onto chick kidney and duck embryo fibroblast cell cultures. In vitro characteristics of Witter's isolates were similar to those reported by Kawamura et al. (1969). The turkey blood and kidney isolates were serologically identical to
Kawamura's isolate. Both turkeys and chickens were susceptible to infection; the virions were shown to be nonpathogenic for both hosts through 8 weeks. Using agar-gel precipitin tests, fluorescent antibody, and neutralization tests Witter et al. (1970) demonstrated a substantial antigenic relationship between HVT and the oncogenic Marek's disease virus (MDV) of chickens. A characteristic of herpesviruses is the ability of the virions to persist for the life of the host, following primary infection (Roizman, 1971). Although the presence is not usually apparent, lesions can periodically occur from which virions can be isolated. One puzzling feature of the herpesviruses is their recurrence in a host possessing antibody to the virions (Roizman, 1971). The importance of herpesviruses as probably causative agents of neoplastic diseases in man and animals has gained increasing attention in recent years. In humans, herpesviruses have been associated with cervical carcinoma (Nahmias, 1969), Burkitt's lymphoma in young children in Africa (Epstein etal, 1964), and postnasal carcinoma occurring in elderly Chinese (Roizman, 1971). Based not only on its well documented lack of pathogenicity, but its efficacy as a vaccine for prevention of Marek's disease, the turkey
494
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
ABSTRACT The in vivo distribution of turkey herpesvirus (HVT) and morphological changes induced by the virus in the visceral organs, blood cells, gonads, and brains of turkeys naturally infected with HVT were examined at the ultrastructural level. Widespread distribution of the virions was found in neural, visceral, and lymphatic tissues. Notable cytoplasmic changes were marked destruction of mitochondria, endoplasmic reticulum, and ribosomes. Nuclear changes included presence of inclusion bodies, clumping and margination of the chromatin, and hypertrophy of the nucleoli. Naked and enveloped virions were seen both in the cytoplasm and nuclei. Cytoplasmic virions were more numerous than nuclear virions. Enveloped virions were of two forms, suggesting different routes of viral envelopment. There was evidence of blood-borne virions in leukocytes in transit through different tissues, and virions were seen in synaptic vesicles within organs suggesting another possible route of infection. Herpes virions in the spleen and intestine were often found in lysosome-like bodies. In leukocytes, vacuoles containing many enveloped virions and myelin figures characteristic of cellular degeneration were often seen. Some cells containing virions had pinocytotic vesicles believed to aid in entry of virions into cells. It was evident that the turkey herpesvirus was capable of infecting numerous tissues often resulting in mild degenerative changes within the cells. 1980 Poultry Science 59:494-499
HERPESVIRUS DISTRIBUTION herpesvirus has had widespread use in vaccination programs for chickens in this country and abroad. No definitive studies of this virus in its natural host, the turkey, have been reported except for histological examination for gross or neoplastic lesions. This study was undertaken because of the close immunological relationship of HVT to the highly oncogenic Marek's disease virus and because of the paucity of information on the distribution and cellular changes induced by the turkey herpesvirus. MATERIALS AND METHODS
RESULTS AND DISCUSSION No bacteria or mycoplasmas were detected in the blood specimens from the test turkeys. Of the tissues examined for HVT infection, the following showed presence of virions: heart, spleen, lung, brain, intestine, kidney, cecal tonsil, proventriculus, bone marrow, white blood cells, and pancreas (Figs. 1—13). Male and female gonadal tissues showed necrotic areas with CPE of herpetic infection, but few virions. No virions were found in liver, gizzard, and muscle specimens although nuclear changes were
seen in the hepatic tissue. It is possible that the virions seen in these tissues could have represented blood-born virions in transit through the organ rather than viral multiplication within the organs. However, the changes noted in tissues containing virions were characteristic of herpes infection. When present, virions were usually found in both the cytoplasm and the nuclei. Changes indicative of viral replication in nuclei consisted of margination of chromatin and presence of nucleocapsids and small nuclear particles approximately 35 nm in diameter. These small nuclear particles were seen in the presence of few virions. Cores of nuclear virions measured 65 nm in diameter and the capsids about 95 nm in diameter. Mature viral particles found in the nuclei measured approximately 165 nm in diameter and each particle consisted of an electron dense core, a capsid, and an envelope. Aggregates of small nuclear particles occurring in crystalline arrangement were seen in several tissues. These particles had diameters of 30 to 40 nm and were associated with capsid-like material. Nuclear inclusions bodies were often seen filling much of the nuclear region and pushing the nuclear chromatin to the periphery. The cytoplasm of infected cells showed severe degeneration of mitochondria, endoplasmic reticulum, and ribosomes. Polykarocytosis, a common occurrence in HVT infected cell cultures, was not seen in this study. In highly degenerative areas, there was some karyolysis and loss of cell membranes, especially in lymphatic tissue. Cytoplasm of the cells in the lymphatic tissue was filled with reticular fibers. In tissue sections of several organs, viral presence was noted with paramyelin figures and in what appeared to be synaptic vesicles. Other herpesviruses have been shown to have a marked predilection for nervous tissue, and virions could spread to other organs through neural routes. The envelopes of virions seen in the cytoplasm seemed to have originated through two different routes. One form of enveloped virion measured 140 to 170 nm in diameter and was seen in smooth-walled vesicles associated with the Golgi complex and the virions were surrounded by a granular substance. Other cytoplasmic enveloped virions measured 200 to 220 nm in diameter and differed from other virions by their triple-layered envelopes. These envelopes
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
Ten 1-year-old Broad-Breasted White Turkeys with natural field infections of HVT were used in this study. Housing and maintenance of turkeys were according to commercial practices. HVT infection in the turkeys was determined by inoculating 24-hr-old primary chick embryo fibroblasts with whole blood taken from the turkey and diluted according to the method of Witter etal. (1970). Blood samples were tested for bacterial contamination using blood agar plates and thioglycollate media at appropriate temperature and incubation periods. Whole blood samples were tested for mycoplasmas using the conventional plate test and also by mixing 1 ml of whole turkey blood with 15 ml of Freye's Media and incubating for 3 days at 37 C and 5 days at 25 C. Liver, spleen, heart, intestine, cecal tonsil, muscle, gizzard, proventriculus, pancreas, kidney, testicles, ovary, brain, lung, bone marrow, and white blood cells were obtained from each turkey for examination. The tissues were processed in 1% osmium tetroxide according to Millonig (1961), embedded in epoxy-araldite mixtures (Mollenhauer, 1963), and stained with lead citrate according to Reynolds (1963).
495
496
HEIN A N D M O R A
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
FIG. FIG. FIG. FIG. FIG.
1. 2. 3. 4. 5.
HVT (V) in splenic sinusoids. X 2 0 , 5 0 0 . H V T in cells adjacent t o spleen capsule. X 2 0 , 5 0 0 . Mature and i m m a t u r e H V T in spleen reticulocyte. X 3 9 , 9 0 0 . Distribution of H V T mainly in fibrillar area of spleen r e t i c u l o c y t e . X 15,400. HVT near d e s m o s o m e (D) and intercellular space in cardiac cell. X 1 7 , 0 0 0 .
HERPESVIRUS DISTRIBUTION
497
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015 FIG. 6. Distorted, tortuous myelinated axons in brain cell. X 10,000. FIG. 7. HVT in cytoplasm of mid brain cell. X 31,300. FIG. 8. HVT in bone marrow granulocyte. X 25,000. FIG. 9. Lymphocyte from circulation containing numerous paramyelin figures (P) and HVT in a vacuole. X 40,000.
498
HEIN AND MORA
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
FIG. FIG. FIG. FIG.
10. Cells of intestine lamina propia with HVT in intercellular space. X 31,900. 11. HVT in lateral interdigitation of adjoining cell processes in cecal tonsil. X 15,900. 12. Distribution of HVT in adjoining dust cells of lung. X 22,000. 13. Pancreatic acinar cell with numerous HVT. X22,500.
499
HERPESVIRUS DISTRIBUTION
Enveloped H V T virions measuring over 2 0 0 n m as reported in this s t u d y have n o t previously been r e p o r t e d in H V T infected cell cultures. T h e largest virions reported here were 1 4 0 t o 1 6 0 n m in d i a m e t e r (Stephens, 1 9 7 2 ; Nazerian etal, 1 9 7 1 ; and Witter etal., 1970). T h e close association of herpesviruses with lysosomes was n o t e d especially in splenic and intestinal tissues. Morphological and biochemical studies with lysosomes suggested possible involvement in intracellular digestion, phagocytosis, pinocytosis, autolysis, and necrosis (DeDuve, 1 9 5 9 ) . F r e e m a n and Grier ( 1 9 6 4 ) correlated t h e findings of paramyelin figures in t h e cytoplasm with cellular degeneration, especially degeneration of cell m e m b r a n e s . This s t u d y s u p p o r t e d t h a t correlation. T h e presence of paramyelin figures c o n c o m i t a n t with H V T infection has n o t b e e n discussed previously except b y Hinshaw ( 1 9 7 3 ) . In l e u k o c y t e s e x a m i n e d in this s t u d y , paramyelin figures were present in b o t h H V T infected cells and cells a p p a r e n t l y free of infection. Data from this s t u d y indicated t h a t t h e r e were similarities b e t w e e n in vivo H V T infection and MDV infection. Both virions appear t o have a m a r k e d affinity for l y m p h a t i c and neural
tissues. T h e d a t a also showed t h a t in spite of t h e wide distribution of herpesvirus in organs and tissues of t u r k e y s with minimal s u b s e q u e n t cellular alterations, t h e infected t u r k e y s did n o t s h o w o u t w a r d signs or s y m p t o m s of herpetic disease.
REFERENCES DeDuve, L., 1959. Lysosomes, a new group of cytoplasmic particles. Page 128 in Subcellular particles. T. Hayashi, ed. Ronald Press, New York. Epstein, M. A., B. G. Achong, and Y. M. Barr. 1964. Viral particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 2 : 7 0 2 - 7 0 3 . Freeman, J. A., and J. C. Grier, 1964. Page 75 in Cellular fine structure. McGraw Hill Book Company, New York. Hinshaw, V. S., 1973. An ultrastructural study of a herpesvirus in peripheral blood cells of the turkey. Ph.D. dissertation, Auburn University, Auburn, AL. Kawamura, H., D. J. King, Jr., and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell cultures of normal turkeys. Avian Dis. 13:853— 862. Millonig, G., 1961. Advantages of a phosphate buffer of OsO„ solutions in fixation. J. Appl. Phys. 32:1637. Mollenhauer, H. H., 1963. Plastic embedding mixture for use in electron microscopy. J. Stain. Rech. 39:111-114. Nahmias, A. J., 1969. Association of genital herpes with cervical cancer. Int. Virology 1:87. Nazerian, K. L., F. Lee, R. L. Witter, and B. R. Burmester, 1971. Ultrastructural studies of a herpesvirus of turkeys antigenically related to Marek's disease virus. Virol. 43:442-452. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17:208-212. Roizman, B., 1971. Herpesvirus, man and cancer or the persistance of the viruses of love. Page 189—214 in Of microbes and life. J. Monod and E. Borek ed. Columbia University Press, New York. Stephens, J., 1972. An ultrastructural study of a herpesvirus of turkeys. Masters thesis, Auburn University, Auburn, AL. Witter, R. L., 1971. Turkey herpesvirus: lack of oncogenicity for turkeys. Avian Dis. 15:666—670. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of a cell associated herpesvirus antigenically related to Marek's disease virus. Amer. J. Vet. Res. 31:525-538. Zygraich, N., and C. Huygelen, 1972. Inoculation of one-day-old chicks with different strains of turkey herpesvirus. II. Virus replication in tissues of inoculated animals. Avian. Dis. 16:793—798.
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
s u r r o u n d virions which seemed t o acquire t h e envelope material at t h e cell m e m b r a n e . In c o m p l e t e virion particles consisting of only nucleocapsids measuring 65 t o 85 n m with n o dense cores were seen in t h e cytoplasm of cells of b o n e m a r r o w , brain, and kidney. F e w extracellular virions were seen associated with cell debris and were non-enveloped; however, t h e H V T virions appeared t o b e highly cell-associated in vivo. In previous histological studies of H V T infected chickens and t u r k e y s , it was f o u n d t h a t H V T did n o t induce neoplasms (Witter, 1 9 7 1 ; and Zygraich and Huygelen ( 1 9 7 2 ) . Ultrastruct u r e analyses of tissues in this s t u d y revealed cytological changes, especially in t h e spleen, b o n e m a r r o w , intestine, cecal tonsil, and brain. Although some l e u k o c y t e s were s h o w n t o contain m a n y enveloped viruses, their presence was n o t accompanied b y t h e m a r k e d cellular changes associated with t h e presence of virions in o t h e r tissues. There was n o a p p a r e n t damage in l e u k o c y t i c cells, n o r loss of nuclear and cellular m e m b r a n e s , as was n o t e d in tissues of o t h e r infected organs. In this s t u d y , few envelo p e d virions were found cell-free.
INTRODUCTION The turkey herpesvirus (HVT) was first isolated from kidney cell cultures of apparently normal turkeys by Kawamura et al. (1969) and the infectious virion was described as being strongly cell associated. Cytopathic effects (CPE) caused by the virion in cell cultures were formation of syncytia, intranuclear inclusions, and small clear plaques. Plaques also were formed on the HVT-inoculated chorio-allantoic membrane of chicken embryos. Inhibition of plaque formation by 5-bromo-2-deoxyuridine suggested the genetic material of the virion to be deoxyribonucleic acid (DNA). Ultrastructural studies revealed that a viral particle contained a dense core measuring 60 nm, a capsid of about 85 x 100 nm, and an envelope 125 X 165 nm in diameter. On the basis of these characteristics, the HVT strain was designated as a Group B herpesvirus (Kawamura etal, 1969; Witter et al, 1970). Witter et al. (1970) isolated a herpesvirus from turkey blood and kidney by inoculating the turkey blood and trypsinized kidney cells onto chick kidney and duck embryo fibroblast cell cultures. In vitro characteristics of Witter's isolates were similar to those reported by Kawamura et al. (1969). The turkey blood and kidney isolates were serologically identical to
Kawamura's isolate. Both turkeys and chickens were susceptible to infection; the virions were shown to be nonpathogenic for both hosts through 8 weeks. Using agar-gel precipitin tests, fluorescent antibody, and neutralization tests Witter et al. (1970) demonstrated a substantial antigenic relationship between HVT and the oncogenic Marek's disease virus (MDV) of chickens. A characteristic of herpesviruses is the ability of the virions to persist for the life of the host, following primary infection (Roizman, 1971). Although the presence is not usually apparent, lesions can periodically occur from which virions can be isolated. One puzzling feature of the herpesviruses is their recurrence in a host possessing antibody to the virions (Roizman, 1971). The importance of herpesviruses as probably causative agents of neoplastic diseases in man and animals has gained increasing attention in recent years. In humans, herpesviruses have been associated with cervical carcinoma (Nahmias, 1969), Burkitt's lymphoma in young children in Africa (Epstein etal, 1964), and postnasal carcinoma occurring in elderly Chinese (Roizman, 1971). Based not only on its well documented lack of pathogenicity, but its efficacy as a vaccine for prevention of Marek's disease, the turkey
494
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
ABSTRACT The in vivo distribution of turkey herpesvirus (HVT) and morphological changes induced by the virus in the visceral organs, blood cells, gonads, and brains of turkeys naturally infected with HVT were examined at the ultrastructural level. Widespread distribution of the virions was found in neural, visceral, and lymphatic tissues. Notable cytoplasmic changes were marked destruction of mitochondria, endoplasmic reticulum, and ribosomes. Nuclear changes included presence of inclusion bodies, clumping and margination of the chromatin, and hypertrophy of the nucleoli. Naked and enveloped virions were seen both in the cytoplasm and nuclei. Cytoplasmic virions were more numerous than nuclear virions. Enveloped virions were of two forms, suggesting different routes of viral envelopment. There was evidence of blood-borne virions in leukocytes in transit through different tissues, and virions were seen in synaptic vesicles within organs suggesting another possible route of infection. Herpes virions in the spleen and intestine were often found in lysosome-like bodies. In leukocytes, vacuoles containing many enveloped virions and myelin figures characteristic of cellular degeneration were often seen. Some cells containing virions had pinocytotic vesicles believed to aid in entry of virions into cells. It was evident that the turkey herpesvirus was capable of infecting numerous tissues often resulting in mild degenerative changes within the cells. 1980 Poultry Science 59:494-499
HERPESVIRUS DISTRIBUTION herpesvirus has had widespread use in vaccination programs for chickens in this country and abroad. No definitive studies of this virus in its natural host, the turkey, have been reported except for histological examination for gross or neoplastic lesions. This study was undertaken because of the close immunological relationship of HVT to the highly oncogenic Marek's disease virus and because of the paucity of information on the distribution and cellular changes induced by the turkey herpesvirus. MATERIALS AND METHODS
RESULTS AND DISCUSSION No bacteria or mycoplasmas were detected in the blood specimens from the test turkeys. Of the tissues examined for HVT infection, the following showed presence of virions: heart, spleen, lung, brain, intestine, kidney, cecal tonsil, proventriculus, bone marrow, white blood cells, and pancreas (Figs. 1—13). Male and female gonadal tissues showed necrotic areas with CPE of herpetic infection, but few virions. No virions were found in liver, gizzard, and muscle specimens although nuclear changes were
seen in the hepatic tissue. It is possible that the virions seen in these tissues could have represented blood-born virions in transit through the organ rather than viral multiplication within the organs. However, the changes noted in tissues containing virions were characteristic of herpes infection. When present, virions were usually found in both the cytoplasm and the nuclei. Changes indicative of viral replication in nuclei consisted of margination of chromatin and presence of nucleocapsids and small nuclear particles approximately 35 nm in diameter. These small nuclear particles were seen in the presence of few virions. Cores of nuclear virions measured 65 nm in diameter and the capsids about 95 nm in diameter. Mature viral particles found in the nuclei measured approximately 165 nm in diameter and each particle consisted of an electron dense core, a capsid, and an envelope. Aggregates of small nuclear particles occurring in crystalline arrangement were seen in several tissues. These particles had diameters of 30 to 40 nm and were associated with capsid-like material. Nuclear inclusions bodies were often seen filling much of the nuclear region and pushing the nuclear chromatin to the periphery. The cytoplasm of infected cells showed severe degeneration of mitochondria, endoplasmic reticulum, and ribosomes. Polykarocytosis, a common occurrence in HVT infected cell cultures, was not seen in this study. In highly degenerative areas, there was some karyolysis and loss of cell membranes, especially in lymphatic tissue. Cytoplasm of the cells in the lymphatic tissue was filled with reticular fibers. In tissue sections of several organs, viral presence was noted with paramyelin figures and in what appeared to be synaptic vesicles. Other herpesviruses have been shown to have a marked predilection for nervous tissue, and virions could spread to other organs through neural routes. The envelopes of virions seen in the cytoplasm seemed to have originated through two different routes. One form of enveloped virion measured 140 to 170 nm in diameter and was seen in smooth-walled vesicles associated with the Golgi complex and the virions were surrounded by a granular substance. Other cytoplasmic enveloped virions measured 200 to 220 nm in diameter and differed from other virions by their triple-layered envelopes. These envelopes
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
Ten 1-year-old Broad-Breasted White Turkeys with natural field infections of HVT were used in this study. Housing and maintenance of turkeys were according to commercial practices. HVT infection in the turkeys was determined by inoculating 24-hr-old primary chick embryo fibroblasts with whole blood taken from the turkey and diluted according to the method of Witter etal. (1970). Blood samples were tested for bacterial contamination using blood agar plates and thioglycollate media at appropriate temperature and incubation periods. Whole blood samples were tested for mycoplasmas using the conventional plate test and also by mixing 1 ml of whole turkey blood with 15 ml of Freye's Media and incubating for 3 days at 37 C and 5 days at 25 C. Liver, spleen, heart, intestine, cecal tonsil, muscle, gizzard, proventriculus, pancreas, kidney, testicles, ovary, brain, lung, bone marrow, and white blood cells were obtained from each turkey for examination. The tissues were processed in 1% osmium tetroxide according to Millonig (1961), embedded in epoxy-araldite mixtures (Mollenhauer, 1963), and stained with lead citrate according to Reynolds (1963).
495
496
HEIN A N D M O R A
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
FIG. FIG. FIG. FIG. FIG.
1. 2. 3. 4. 5.
HVT (V) in splenic sinusoids. X 2 0 , 5 0 0 . H V T in cells adjacent t o spleen capsule. X 2 0 , 5 0 0 . Mature and i m m a t u r e H V T in spleen reticulocyte. X 3 9 , 9 0 0 . Distribution of H V T mainly in fibrillar area of spleen r e t i c u l o c y t e . X 15,400. HVT near d e s m o s o m e (D) and intercellular space in cardiac cell. X 1 7 , 0 0 0 .
HERPESVIRUS DISTRIBUTION
497
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015 FIG. 6. Distorted, tortuous myelinated axons in brain cell. X 10,000. FIG. 7. HVT in cytoplasm of mid brain cell. X 31,300. FIG. 8. HVT in bone marrow granulocyte. X 25,000. FIG. 9. Lymphocyte from circulation containing numerous paramyelin figures (P) and HVT in a vacuole. X 40,000.
498
HEIN AND MORA
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
FIG. FIG. FIG. FIG.
10. Cells of intestine lamina propia with HVT in intercellular space. X 31,900. 11. HVT in lateral interdigitation of adjoining cell processes in cecal tonsil. X 15,900. 12. Distribution of HVT in adjoining dust cells of lung. X 22,000. 13. Pancreatic acinar cell with numerous HVT. X22,500.
499
HERPESVIRUS DISTRIBUTION
Enveloped H V T virions measuring over 2 0 0 n m as reported in this s t u d y have n o t previously been r e p o r t e d in H V T infected cell cultures. T h e largest virions reported here were 1 4 0 t o 1 6 0 n m in d i a m e t e r (Stephens, 1 9 7 2 ; Nazerian etal, 1 9 7 1 ; and Witter etal., 1970). T h e close association of herpesviruses with lysosomes was n o t e d especially in splenic and intestinal tissues. Morphological and biochemical studies with lysosomes suggested possible involvement in intracellular digestion, phagocytosis, pinocytosis, autolysis, and necrosis (DeDuve, 1 9 5 9 ) . F r e e m a n and Grier ( 1 9 6 4 ) correlated t h e findings of paramyelin figures in t h e cytoplasm with cellular degeneration, especially degeneration of cell m e m b r a n e s . This s t u d y s u p p o r t e d t h a t correlation. T h e presence of paramyelin figures c o n c o m i t a n t with H V T infection has n o t b e e n discussed previously except b y Hinshaw ( 1 9 7 3 ) . In l e u k o c y t e s e x a m i n e d in this s t u d y , paramyelin figures were present in b o t h H V T infected cells and cells a p p a r e n t l y free of infection. Data from this s t u d y indicated t h a t t h e r e were similarities b e t w e e n in vivo H V T infection and MDV infection. Both virions appear t o have a m a r k e d affinity for l y m p h a t i c and neural
tissues. T h e d a t a also showed t h a t in spite of t h e wide distribution of herpesvirus in organs and tissues of t u r k e y s with minimal s u b s e q u e n t cellular alterations, t h e infected t u r k e y s did n o t s h o w o u t w a r d signs or s y m p t o m s of herpetic disease.
REFERENCES DeDuve, L., 1959. Lysosomes, a new group of cytoplasmic particles. Page 128 in Subcellular particles. T. Hayashi, ed. Ronald Press, New York. Epstein, M. A., B. G. Achong, and Y. M. Barr. 1964. Viral particles in cultured lymphoblasts from Burkitt's lymphoma. Lancet 2 : 7 0 2 - 7 0 3 . Freeman, J. A., and J. C. Grier, 1964. Page 75 in Cellular fine structure. McGraw Hill Book Company, New York. Hinshaw, V. S., 1973. An ultrastructural study of a herpesvirus in peripheral blood cells of the turkey. Ph.D. dissertation, Auburn University, Auburn, AL. Kawamura, H., D. J. King, Jr., and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell cultures of normal turkeys. Avian Dis. 13:853— 862. Millonig, G., 1961. Advantages of a phosphate buffer of OsO„ solutions in fixation. J. Appl. Phys. 32:1637. Mollenhauer, H. H., 1963. Plastic embedding mixture for use in electron microscopy. J. Stain. Rech. 39:111-114. Nahmias, A. J., 1969. Association of genital herpes with cervical cancer. Int. Virology 1:87. Nazerian, K. L., F. Lee, R. L. Witter, and B. R. Burmester, 1971. Ultrastructural studies of a herpesvirus of turkeys antigenically related to Marek's disease virus. Virol. 43:442-452. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17:208-212. Roizman, B., 1971. Herpesvirus, man and cancer or the persistance of the viruses of love. Page 189—214 in Of microbes and life. J. Monod and E. Borek ed. Columbia University Press, New York. Stephens, J., 1972. An ultrastructural study of a herpesvirus of turkeys. Masters thesis, Auburn University, Auburn, AL. Witter, R. L., 1971. Turkey herpesvirus: lack of oncogenicity for turkeys. Avian Dis. 15:666—670. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of a cell associated herpesvirus antigenically related to Marek's disease virus. Amer. J. Vet. Res. 31:525-538. Zygraich, N., and C. Huygelen, 1972. Inoculation of one-day-old chicks with different strains of turkey herpesvirus. II. Virus replication in tissues of inoculated animals. Avian. Dis. 16:793—798.
Downloaded from http://ps.oxfordjournals.org/ at University of Michigan on June 13, 2015
s u r r o u n d virions which seemed t o acquire t h e envelope material at t h e cell m e m b r a n e . In c o m p l e t e virion particles consisting of only nucleocapsids measuring 65 t o 85 n m with n o dense cores were seen in t h e cytoplasm of cells of b o n e m a r r o w , brain, and kidney. F e w extracellular virions were seen associated with cell debris and were non-enveloped; however, t h e H V T virions appeared t o b e highly cell-associated in vivo. In previous histological studies of H V T infected chickens and t u r k e y s , it was f o u n d t h a t H V T did n o t induce neoplasms (Witter, 1 9 7 1 ; and Zygraich and Huygelen ( 1 9 7 2 ) . Ultrastruct u r e analyses of tissues in this s t u d y revealed cytological changes, especially in t h e spleen, b o n e m a r r o w , intestine, cecal tonsil, and brain. Although some l e u k o c y t e s were s h o w n t o contain m a n y enveloped viruses, their presence was n o t accompanied b y t h e m a r k e d cellular changes associated with t h e presence of virions in o t h e r tissues. There was n o a p p a r e n t damage in l e u k o c y t i c cells, n o r loss of nuclear and cellular m e m b r a n e s , as was n o t e d in tissues of o t h e r infected organs. In this s t u d y , few envelo p e d virions were found cell-free.