Replication of Herpesvirus of Turkeys in Chick Fibroblasts JOYCE STEPHENS AND E .
C.
MORA
Department of Poultry Science, Alabama Agricultural Experiment Station, Auburn University, Alabama 36830
Auburn,
(Received for publication May 13, 1975)
POULTRY SCIENCE 55: 286-295, 1976
INTRODUCTION
P
ROGRESS is being made in the etiology and control of malignancies in man as well as other forms of animal life. A herpesvirus (HVT) recently isolated from the blood turkeys is of particular importance because it has been shown to be an effective biological agent in the control of certain types of Marek's disease of chickens. The mechanism of protection by HVT is not yet understood. A comparative study of the work of many authors on the herpes group of viruses was made by Plummer (1967), who made reference to work with some herpesviruses which suggested that the infective capacity was associated with the envelopment of the viruses; however, the requirement for envelopment is not conclusive because of evidence of absorption of some naked viruses. Melnick et al. (1964) stated that viruses of the herpes group appear to fall into two subgroups, designated A and B, based on
their behavior in tissue culture. Group A consisted of viruses which are readily released from cells in an active form and group B as viruses which are cell-associated. Herpesviruses produce distinct characteristics when grown in cultures of suitable cells of their natural hosts. The cytopathic effects vary with species and with strains of herpesviruses and appear to progress from simple rounding of the cells through "ballooning" of individual cells to the formation of syncytia which vary from a few nuclei to many hundreds of nuclei in a given mass of cytoplasm. Some infected cells stained with hematoxylin/eosin revealed type A intranuclear inclusions which were eosinophilic and centrally located and the inclusions did not represent "colonies" of viruses but reflected areas of viral DNA synthesis. The host cell chromatin is usually scattered and appears as a margin at the peripheral part of the nucleus (Plummer, 1967). A virus possessing some of the charac286
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ABSTRACT An ultrastructural study of chick embryo fibroblasts infected with herpesvirus of turkeys (HVT) revealed that infection produced degenerative effects in the cells ranging from alteration of cellular structures to complete lysis of the cells. Infection was indicated by margination of the nuclear chromatin, the presence of nuclear and cytoplasmic virions, destruction of mitochondria, loss of rough and smooth endoplasmic reticulum, and polykaryocytosis. Viruses entered the cell by penetrating the cytoplasmic membrane, and replication took place within the nucleus. Small nuclear particles observed as aggregates in the presence of few herpesvirions appeared to be a part of the replication of the virus. Viruses were released from the cell nucleus either naked or enveloped. Where karyolysis occurred, naked viruses were released as the karyoplasm diffused into the cytoplasm. Enveloped viruses were not observed leaving the nucleus, but it appeared that departure could have occurred by the budding of the viruses through the nuclear membrane. Where complete lysis of the cells occurred, the naked viruses were released into the extracellular fluid. Enveloped viruses were observed only in intact cells. Viruses which were observed extracellularly were naked viruses, and those that were observed entering the cell by penetrating the cytoplasmic membrane were also naked. This evidence suggests that in vitro HVT does not require the envelope to be infective.
HERPESVIRUS IN FIBROBLASTS
cytoplasmic bridges which perhaps led to the eventual fusion of infected cells. In the polykarocytes with many nuclei, the cell membranes were usually disintegrated and the cytoplasmic structures were drastically changed. The cytoplasm was highly vacuolated, and normal components of the cell such as mitochondria, endoplasmic reticulum, and ribosomes were either absent or damaged and disorganized. Naked virions were observed in some nuclei. Since current immunological hypotheses do not satisfactorily explain the mechanism of protection, other undescribed mechanisms may be involved. One possibility is that the vaccine virus and the host cell DNA block a similar interaction that would have occurred between the Marek's disease virus and the cell, thus preventing the disease. The development of the anti-Marek's disease vaccine and the understanding of its mechanism of action will have far-reaching implications in oncogenic virology and the avian system can be used as a model to help understand the etiology of some malignancies and to develop means for the control of some virus induced cancers in animals. MATERIALS AND METHODS Chick embryo fibroblasts were prepared from 8-day-old embryos of an AuburnDryden Leghorn strain. The embryos were minced and washed twice in Earle's balanced salt solution (BSS), then suspended in .25% trypsin at 37° C. for 40 minutes. The trypsin was removed by centrifugation and washing the cells three times in BSS, after which the cells were suspended in Eagle's MEM growth medium and 25 cm. 2 Falcon tissue culture flasks were seeded with .25 ml. each of the fibroblast suspension. After 24 hours incubation at 37° C. monolayers were formed and the growth medium was replaced with maintenance medium. After 48 hours incubation in maintenance
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teristics of herpesvirus was isolated from turkeys and grown in cell cultures (Witter et al., 1970). This herpesvirus of the turkey (HVT) was shown to be antigenically related to the Marek's disease herpesvirus (MDHV) (Witter et al, 1970) and to be an effective agent for protecting chickens from the development of malignant tumors caused by MDHV (Purchase and Okazaki, 1971; Purchase et al, 1971b, c; Okazaki et al, 1970; Eidson and Anderson, 1971; and Eidson et al, 1971). HVT was found to be apparently nonpathogenic for chickens at least through 20 weeks of age and was usually not transmitted by contact in chickens (Okazaki et al., 1970). Purchase et al. (1971a) found that infection in the different cell cultures was similar and was characterized by plaques of spherical, highly refractile cells, which in some cases, became detached from the monolayer producing a central clear area. Eosinophilic Cowdry type A intranuclear inclusions were also observed in several types of cell cultures. Mustaffa-Babjee et al (1971), Witter et al. (1970), and Kawamura et al. (1969) reported similar results for the development of characteristic cytopathic effects and presented evidence for HVT being a cell-associated virus. Characteristics of infected cells and the ultrastructure of HVT have been studied using electron microscopy (Nazerian et al, 1971; and Witter et al, 1970). The study by Nazerian et al. (1971) indicated that one of the prominent features of cell cultures infected with HVT was formation of polykaryocytes which varied from few to many nuclei. In cells with few nuclei, the ctyoplasmic membrane was usually intact, however, there were changes related to rival replication such as margination of the nuclear chromatin and the presence of viral nucleocapsides and small nuclear particles 35 nm. in diameter. The opposing membranes of some infected neighboring cells were observed in the process of lysis, allowing the formation of many
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All electron micrographs are of infected chick embryo fibroblasts four days postinoculation. 1. Virus (V) penetrating a cell. The virus appears to have a dense core and its surface is covered with granular material. The virus is naked and measured 90 nm. in diameter. 60,420x. 2. Virus (V) penetrating a cell. This is a naked virus with a dense core and measures 96 nm. in diameter. 79,800x. 3. Infection is indicated in the cell by margination of the nuclear chromatin (C) and the presence of nuclear viruses (V). Mitochondria (M). rough endoplasmic reticulum (R), and a Golgi (G) are also present. 10,260x. 4. The cell contains several large vacuoles (Vac) that have distorted the shape of the nucleus (N). The nuclear chromatin is marginated and there are viruses within the nucleus. 6.840x. 5. Evidence of karyolysis. The karyoplasm and cytoplasm have fused and several naked virions (V) were released into the cytoplasm. Several structures which appear to be mitochondria (M) are also present. 15,960x.
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6. Evidence of lysis of a cell. Numerous naked virions are being released into the extracellular space. No distinguishable organelles are present. 15,960x. 7. A polykaryocyte containing two nuclei (N). Both nuclei contain virus particles. There is some vacuolation and loss of cytoplasmic organelles. 8,094x. 8. Aggregate of nuclear particles (P). Herpesvirions (V) each containing four visible electron dense particles are seen associated with the aggregate. 31,920x. 9. Intranuclear viruses containing electron dense particles arranged so that the center of the virus has the appearance of an electron lucent cross. Several nuclear particles (P) are also present. The chromatin is marginated. 51.300x.
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10. Intranuclear viruses. The naked viruses (V) which measure 108 nm. in diameter have electron dense cores, contain several electron dense particles, or are empty. The enveloped viruses (E) measure 144 nm. in diameter and are located within a nuclear vesicle. 31,920x. 11. Enveloped and naked nuclear viruses within a vesicle. Some of the enveloped viruses are empty and some contain electron dense particles. 51,300x. 12. Two cytoplasmic enveloped viruses that are similar to nuclear enveloped viruses. A nuclear virus is also seen. 39,900x. 13. Enveloped, cytoplasmic viruses (V) measuring 144 nm. in diameter. One virus appears to be budding from a cytoplasmic vacuole (Vac). Cytoplasmic organelles are indistinguishable. One enveloped virus is in the extracellular spaces. 39,9O0x.
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14. Cytoplasmic enveloped viruses. These viruses are similar to nuclear enveloped viruses. 51,300x. 15. The viruses in the extracellular space appear to have budded from the cytoplasmic membrane as is indicated by the projection of the membrane. The viruses have electron dense cores and measure 96 nm. in diameter. 39,900x. 16. Naked viruses (V) being released into the extracellular space as a result of lysis of the cell. Two other structures which resemble disorganized mitochondria (M) are also present. 31.920x. 17. Extracellular viruses closely associated with an intact cytoplasmic membrane. The viruses are naked, have dense cores, and measure 96 nm. in diameter. 39,900x.
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To embed the cells in 100% Epoxy mixture, the pellets were cut into small pieces and placed in the tips of capsules containing a small amount of embedding medium. After the specimens were positioned, capsules were filled and kept at 60° C. for 48 hours for polymerization of the plastic. Ultrathin sections (600 to 700 A. thick) were stained using 2% aqueous uranly acetate (Huxley et al., 1961) for 40 minutes at 60° C. followed by 8 minutes in lead citrate (Reynolds, 1963) at room temperature.
RESULTS AND DISCUSSION In the noninfected cell culture, the presence of herpesvirions was not observed nor was there evidence of any viral infection. The cells were intact, lying adjacent to each other but separated by their cytoplasmic membranes. There was no evidence of cell separation nor degeneration. The nuclei were circular to ovoid and usually occupied a portion of the cell toward one end. The membranes of the nuclei were intact, and the chromatin appeared as a centrally located electron dense mass. The most distinct structures of the cytoplasm were the mitochondria, the rough and smooth endoplasmic reticulum, and masses of polyribosomes. The mitochondria were enclosed by a double layered membrane and contained an organized internal arrangement of cristae. Networks of rough and smooth endoplasmic reticulum were extensive throughout the cytoplasm. In the cell cultures infected with HVT, viral infectivity of ten produced only slight changes in the nuclei and in the cytoplasm; however, in other cells there was complete degeneration with loss of the cytoplasmic and nuclear membranes. In cells in which the membranes were still intact, there was often extensive vacuolation of the cytoplasm which caused distortion of the nuclei. There was limited cytoplasmic space available and marked loss and degeneration of cytoplasmic organelles. There was margination of the nuclear chromatin and usually the presence of several to many herpesvirions could be found scattered in the nuclei and in the cytoplasm. In these cells, the mitochondria appeared to have lost their internal arrangement of cristae and to have decreased in size. The extensive network of rough and smooth endoplasmic reticulum was lost, and only fragments of mostly rough endoplasmic reticulum remained. The presence of aggregates of small nuclear particles was observed in some cells. These particles were found in the presence
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medium 4.5 ml. of the culture fluid were removed from each of the flasks to increase the probability of virus-cell contact and each flask was inoculated with .2 ml. of the HVT virus stock (Merck, Deptavac-HVT). After 3 hours incubation, 4.0 ml. of maintenance medium were added to each flask. Twenty four hours postinoculation the medium was removed from each flask and replaced with new maintenance medium. The cell cultures were examined daily with the light microscope for the development of cytopathic effects (CPE). Four days postinoculation noticeable CPE was observed in the infected flasks and the monolayers were detached from the flasks by a gentle rotation of the flasks. The monolayers from the flasks were pooled and centrifuged in a clinical centrifuge to form a cell pellet. The monolayers from the control flasks were removed with trypsin, pooled and centrifuged. The pellets were suspended in 1% osmium tetroxide prepared according to Millonig (1961) and fixed for 1 hour at 4° C , then dehydrated through graded alcohols and propylene oxide according to traditional manner for epoxy embedment and embedded in an Epon-Araldite mixture composed of two parts of solution A (62 ml. Araldite 506 and 100 ml. dodecenyl succinic anhydride), one part solution B (100 ml. Araldite 506 and 89 ml. nadic methyl anahydride) and 1 / 10th volume Epon 812 with 1.5-2% tris dimethyl aminomethyl phenol-30 as catalyst.
HERPESVIRUS IN FIBROBLASTS
Herpesvirions were observed in all cells in which there were indications of cellular destruction. Most of the virus particles were seen in the nuclei; however, occasionally, viruses were seen in the cytoplasm. In cells containing crystals of nuclear particles, the predominant type of virus was one that contained several electron dense particles which often were arranged so that the center of the virus appeared as an electron lucent cross. This type of virus also occurred in the presence of other types of virions, in the absence of any nuclear particles, and as both a naked and enveloped virus. Other types of naked viruses were those with electron dense cores and those in which the capsid was empty. Naked viruses measured 95 to 110 nm. in diameter.
Enveloped viruses present in the nucleus and cytoplasm were observed only in intact cells. Envelopment apparently occurred by the budding of viruses either into or out of vacuoles. In the nuclei containing both naked and enveloped viruses, the enveloped viruses occurred only within vesicles. In the cytoplasm, the enveloped viruses were seen both free of any enclosing vacuole and budding from a vacuole. Enveloped viruses measured 140 to 160 nm. in diameter. Only naked virions were observed in cells in which karyolysis was complete and in which the cytoplasmic membranes were lost. Naked virions of the three types described previously were observed in the remaining nuclear material; however, viruses in the cytoplasm usually had electron dense cores and were surrounded by granular material. Extracellular viruses associated with cell debris were quite numerous and measured 95 to 100 nm. in diameter, had electron dense cores, were unenveloped, and were usually masked by the deposition of granular material. Viruses were observed in the process of budding both into and out of the cytoplasmic membranes. Viruses budding from the membrane averaged 115 nm. in diameter, had electron dense cores, and were not enveloped. Viruses budding into the cell were unenveloped and averaged 106 nm. in diameter. Viruses were often found closely associated with the cytoplasmic membrane, but were rarely budding. In many cells in which there were few indications of viral infection other than margination of the chromatin, the nuclei contained aggregates of crystals of nuclear particles. The particles measured 35 nm. in diameter, had translucent centers, and were bordered by electron dense material. Aggregates of particles usually occurred at the periphery of the nuclei, and as many as three to four crystals were found in a single nucleus. In nuclei containing the crystals, very few
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of only limited numbers of virions and were located at the periphery of the nuclei. With progression of infection, degenerative effects of the cells were more evident. Gradually, karyolysis occurred and the karyoplasm diffused into the cytoplasm. The nuclear chromatin no longer formed a marginal rim at the periphery of the nucleus but occurred as electron dense clumps intermingled with cytoplasm. Further destruction of mitochondria was characterized by complete loss of internal and external integrity. Other cytoplasmic organelles were lost as the cells progressed to complete disorganization. There was release of naked virions from the nuclear area into the cytoplasm and intercellular spaces due to the focal dissolution of the cytoplasmic membrane. Infection resulted in the formation of polykaryocytes which usually contained two to three nuclei. The absence of distinct cytoplasmic membranes between adjacent cells suggested fusion of the cells resulting in a multinucleated mass of cytoplasm. This characteristic was not observed in control cell cultures. The morphological changes produced by viral infection were similar for polykaryocytes and mononucleated cells.
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The infectivity of the two enveloped viruses has not been determined. Nazerian et al. (1971) suggested that infectivity could be similar to the herpes simplex virions in which the bulk of the infectivity is associated with the cytoplasmic fraction. In a study by Nii et al. (1968) of the sequence of the
development of the group A, herpes simplex virus (HSV), it was concluded that the unenveloped capsid is unstable outside the cell and that the enveloped capsids are the infectious or complete viruses. The mechanism of infectivity of HVT is debatable. There was evidence to suggest that HVT can leave the cell by budding through the cytoplasmic membrane. This was not a common observation and the viruses involved were naked viruses. Extracellular viruses were, however, numerous. Many cells were seen in the process of lysis resulting in the loss of the cytoplasmic membrane with the release of viruses into the extracellular fluid. Aggregates of viruses were found both associated with cell debris far removed from any intact cells and also closely associated with the cytoplasmic membranes of intact cells. The release of viruses from cells did appear to be by rupture of cells rather than by budding from intact cells. With HVT infection, numerous naked viruses were found extracellularly and also budding into cells. This evidence indicates that in vitro HVT does not require the envelope to be infective and that the naked extracellular viruses are mature, functionally infective viruses. REFERENCES Eidson,C.S.,andD. P.Anderson, 1971. Immunization against Marek's disease. Avian Dis. 15: 68-81. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15: 312-322. Epstein, M. A., B. G. Achong, A. E. Churchill and P. M. Biggs, 1968. Structure and development of the herpes-type virus of Marek's disease. U.S. National Cancer Institute J. 41: 805-820. Gravell, M., A. Granoff and R. W. Darlington, 1968. Viruses and renal carcinoma of Rana pipiens, VII propagation of a herpestype frog virus. Virology, 36: 467-475. Huxley, H. E., and G. Zubay, 1961. Preferential staining of nucleic acid-containing structures for electron microscopy. J. Biophysic. Biochem. Cytol. 11: 273-296.
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herpesvirions were present. The viruses observed usually contained either three to four electron dense particles at the periphery of the virion or one centrally located particle. In both types, the enclosed particles were morphologically identical to the nuclear particles. Cells with many herpesvirions did not contain aggregates of the nuclear particles. Viruses in which the capsid was empty might have appeared so due to the particular section cut. Sectioning and the position of the virus could also account for those viruses containing only electron dense particle. As for those viruses having extremely dense cores, it was not possible to determine whether they were primary forms of the virus or perhaps a mature form of those viruses containing several particles, having been formed by the condensation of the particles into one dense mass. Since both forms of the virus have been seen enveloped, this suggestion of maturation seems unlikely. In the nucleus, viruses appeared to acquire the envelope either by budding into nuclear vesicles or by budding through the nuclear membrane into the cytoplasm and the envelope had the appearance of a membrane surrounding the capsid and was somewhat granular. The majority of the nuclear enveloped viruses had electron dense cores. Envelopment in the cytoplasm appeared to occur as viruses budded out of cytoplasmic vacuoles and two types of enveloped were seen. One type was similar to nuclear enveloped viruses and a second type of envelope appeared to be concentrically layered about the capsid which enclosed a moderately electron dense core. The enveloped viruses measured 140 to 160 nm. in diameter.
HERPESVIRUS IN FIBROBLASTS
Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413-429. Plummer, G., 1967. Comparative virology of the herpes group. Progr. Med. Virol. 9: 302-340. Purchase, H. G., B. R. Burmester and C. H. Cunningham, 1971a. Responses of cell cultures from various avian species to Marek's disease virus and herpesvirus of turkeys. Amer. J. Vet. Res. 32: 1811-1823. Purchase, H. G., and W. Okazaki, 1971. Effect of vaccination with herpesvirus of turkeys (HVT) on horizontal spread of Marek's disease herpesvirus. Avian Dis. 15: 391-397. Purchase, H. G., W. Okazaki and B. R. Burmester, 1971b. Field trials with the herpes virus of turkeys (HVT) strain FC 126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Purchase, H. G., R. L. Witter, W. Okazaki and B. R. Burmester, 1971c. Vaccination against Marek's disease, In: Perspectives in Virology, (Gustav Stern Symposium), edited by M. Pollard, Academic Press, New York, New York, vol. 7, p. 91-110. 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. 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.
NEWS AND NOTES (Continued from page 273) Manufacturing Division, Merck Animal Health Division, and Merck Chemical Division. This reorganization accompanied major changes in executive responsibility and organizational alignment that Merck President John J. Horan said would enhance the company's ability to meet future challenges and opportunities. Dr. Hilmer L. Jones has been elected Vice President and General Manager of the Merck Animal Health Division, formerly the Animal Health and Feed Products area. Dr. Jones had been appointed General Manager of the Animal Health and Feed Products Department in October, 1974, after joining Merck in 1960 as Director of Technical Services for that Depart-
ment. He previously had served in executive positions in the agricultural and veterinary field with Pfizer, Inc. A native of Crenshaw County, Alabama, Dr. Jones received a B.S. degree, majoring in agricultural science, an M.S. degree, majoring in animal nutrition, and a D.V.M. degree at Auburn University. He formerly was Chairman of the Division of Environmental Health, Auburn University Extension Service. He helped organize and develop the Division. He also has been involved in several voluntary activities with veterinary and health planning organizations. In his new position, Dr. Jones reports to John L. Huck, Senior Vice President of Merck & Co., Inc.
(Continued on page 324)
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Kaleta, E. F., and R. A. Bankowski, 1972a. Production of circulating and cell-broud interferon in chickens by type 1 and type 2 plaque-producing agents of the Cal-1 strain of Marek's disease herpesvirus and herpesvirus of turkeys. Amer. J. Vet. Res. 33: 567-571. Kaleta, E. F. and R. A. Bankowski, 1972b. Production of interferon by the Cal-1 and turkey herpesvirus strains associated with Marek's disease. Amer. J. Vet. Res. 33: 573-577. Kawamura, H. G., J. King, Jr. and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell cultures of normal turkeys. Avian Dis. 13: 853-862. Melnick, J. L., M. Midulla, I. Wimberly, J. G. Barrera-Oro and B. M. Levy, 1964. A new member of the herpesvirus group isolated from South American marmosets. J. Immunol. 92: 596-601. Millonig, G., 1961. Advantages of a phosphate buffer for O s 0 4 solutions in fixation. J. Appl. Phys. ew: 1637. Mustaffa-Babjee, A., P. Ketterer and P. B. Spradbrow, 1971. Isolation of turkey herpesvirus in cell culture. Aust. Vet. J. 47: 125. Nazerian, K., and R. L. Witter, 1970. Cell-free transmission and in vivo replication of Marek's disease virus. J. Virol. 5: 388-397. 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. Virology, 43: 442-452. Nii, S. Hiro, C. Morgan and H. M. Rose, 1968. Electron microscopy of herpes simplex virus, II Sequence of development. J. Virol. 2: 517-536.
295
C.
MORA
Department of Poultry Science, Alabama Agricultural Experiment Station, Auburn University, Alabama 36830
Auburn,
(Received for publication May 13, 1975)
POULTRY SCIENCE 55: 286-295, 1976
INTRODUCTION
P
ROGRESS is being made in the etiology and control of malignancies in man as well as other forms of animal life. A herpesvirus (HVT) recently isolated from the blood turkeys is of particular importance because it has been shown to be an effective biological agent in the control of certain types of Marek's disease of chickens. The mechanism of protection by HVT is not yet understood. A comparative study of the work of many authors on the herpes group of viruses was made by Plummer (1967), who made reference to work with some herpesviruses which suggested that the infective capacity was associated with the envelopment of the viruses; however, the requirement for envelopment is not conclusive because of evidence of absorption of some naked viruses. Melnick et al. (1964) stated that viruses of the herpes group appear to fall into two subgroups, designated A and B, based on
their behavior in tissue culture. Group A consisted of viruses which are readily released from cells in an active form and group B as viruses which are cell-associated. Herpesviruses produce distinct characteristics when grown in cultures of suitable cells of their natural hosts. The cytopathic effects vary with species and with strains of herpesviruses and appear to progress from simple rounding of the cells through "ballooning" of individual cells to the formation of syncytia which vary from a few nuclei to many hundreds of nuclei in a given mass of cytoplasm. Some infected cells stained with hematoxylin/eosin revealed type A intranuclear inclusions which were eosinophilic and centrally located and the inclusions did not represent "colonies" of viruses but reflected areas of viral DNA synthesis. The host cell chromatin is usually scattered and appears as a margin at the peripheral part of the nucleus (Plummer, 1967). A virus possessing some of the charac286
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ABSTRACT An ultrastructural study of chick embryo fibroblasts infected with herpesvirus of turkeys (HVT) revealed that infection produced degenerative effects in the cells ranging from alteration of cellular structures to complete lysis of the cells. Infection was indicated by margination of the nuclear chromatin, the presence of nuclear and cytoplasmic virions, destruction of mitochondria, loss of rough and smooth endoplasmic reticulum, and polykaryocytosis. Viruses entered the cell by penetrating the cytoplasmic membrane, and replication took place within the nucleus. Small nuclear particles observed as aggregates in the presence of few herpesvirions appeared to be a part of the replication of the virus. Viruses were released from the cell nucleus either naked or enveloped. Where karyolysis occurred, naked viruses were released as the karyoplasm diffused into the cytoplasm. Enveloped viruses were not observed leaving the nucleus, but it appeared that departure could have occurred by the budding of the viruses through the nuclear membrane. Where complete lysis of the cells occurred, the naked viruses were released into the extracellular fluid. Enveloped viruses were observed only in intact cells. Viruses which were observed extracellularly were naked viruses, and those that were observed entering the cell by penetrating the cytoplasmic membrane were also naked. This evidence suggests that in vitro HVT does not require the envelope to be infective.
HERPESVIRUS IN FIBROBLASTS
cytoplasmic bridges which perhaps led to the eventual fusion of infected cells. In the polykarocytes with many nuclei, the cell membranes were usually disintegrated and the cytoplasmic structures were drastically changed. The cytoplasm was highly vacuolated, and normal components of the cell such as mitochondria, endoplasmic reticulum, and ribosomes were either absent or damaged and disorganized. Naked virions were observed in some nuclei. Since current immunological hypotheses do not satisfactorily explain the mechanism of protection, other undescribed mechanisms may be involved. One possibility is that the vaccine virus and the host cell DNA block a similar interaction that would have occurred between the Marek's disease virus and the cell, thus preventing the disease. The development of the anti-Marek's disease vaccine and the understanding of its mechanism of action will have far-reaching implications in oncogenic virology and the avian system can be used as a model to help understand the etiology of some malignancies and to develop means for the control of some virus induced cancers in animals. MATERIALS AND METHODS Chick embryo fibroblasts were prepared from 8-day-old embryos of an AuburnDryden Leghorn strain. The embryos were minced and washed twice in Earle's balanced salt solution (BSS), then suspended in .25% trypsin at 37° C. for 40 minutes. The trypsin was removed by centrifugation and washing the cells three times in BSS, after which the cells were suspended in Eagle's MEM growth medium and 25 cm. 2 Falcon tissue culture flasks were seeded with .25 ml. each of the fibroblast suspension. After 24 hours incubation at 37° C. monolayers were formed and the growth medium was replaced with maintenance medium. After 48 hours incubation in maintenance
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teristics of herpesvirus was isolated from turkeys and grown in cell cultures (Witter et al., 1970). This herpesvirus of the turkey (HVT) was shown to be antigenically related to the Marek's disease herpesvirus (MDHV) (Witter et al, 1970) and to be an effective agent for protecting chickens from the development of malignant tumors caused by MDHV (Purchase and Okazaki, 1971; Purchase et al, 1971b, c; Okazaki et al, 1970; Eidson and Anderson, 1971; and Eidson et al, 1971). HVT was found to be apparently nonpathogenic for chickens at least through 20 weeks of age and was usually not transmitted by contact in chickens (Okazaki et al., 1970). Purchase et al. (1971a) found that infection in the different cell cultures was similar and was characterized by plaques of spherical, highly refractile cells, which in some cases, became detached from the monolayer producing a central clear area. Eosinophilic Cowdry type A intranuclear inclusions were also observed in several types of cell cultures. Mustaffa-Babjee et al (1971), Witter et al. (1970), and Kawamura et al. (1969) reported similar results for the development of characteristic cytopathic effects and presented evidence for HVT being a cell-associated virus. Characteristics of infected cells and the ultrastructure of HVT have been studied using electron microscopy (Nazerian et al, 1971; and Witter et al, 1970). The study by Nazerian et al. (1971) indicated that one of the prominent features of cell cultures infected with HVT was formation of polykaryocytes which varied from few to many nuclei. In cells with few nuclei, the ctyoplasmic membrane was usually intact, however, there were changes related to rival replication such as margination of the nuclear chromatin and the presence of viral nucleocapsides and small nuclear particles 35 nm. in diameter. The opposing membranes of some infected neighboring cells were observed in the process of lysis, allowing the formation of many
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All electron micrographs are of infected chick embryo fibroblasts four days postinoculation. 1. Virus (V) penetrating a cell. The virus appears to have a dense core and its surface is covered with granular material. The virus is naked and measured 90 nm. in diameter. 60,420x. 2. Virus (V) penetrating a cell. This is a naked virus with a dense core and measures 96 nm. in diameter. 79,800x. 3. Infection is indicated in the cell by margination of the nuclear chromatin (C) and the presence of nuclear viruses (V). Mitochondria (M). rough endoplasmic reticulum (R), and a Golgi (G) are also present. 10,260x. 4. The cell contains several large vacuoles (Vac) that have distorted the shape of the nucleus (N). The nuclear chromatin is marginated and there are viruses within the nucleus. 6.840x. 5. Evidence of karyolysis. The karyoplasm and cytoplasm have fused and several naked virions (V) were released into the cytoplasm. Several structures which appear to be mitochondria (M) are also present. 15,960x.
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6. Evidence of lysis of a cell. Numerous naked virions are being released into the extracellular space. No distinguishable organelles are present. 15,960x. 7. A polykaryocyte containing two nuclei (N). Both nuclei contain virus particles. There is some vacuolation and loss of cytoplasmic organelles. 8,094x. 8. Aggregate of nuclear particles (P). Herpesvirions (V) each containing four visible electron dense particles are seen associated with the aggregate. 31,920x. 9. Intranuclear viruses containing electron dense particles arranged so that the center of the virus has the appearance of an electron lucent cross. Several nuclear particles (P) are also present. The chromatin is marginated. 51.300x.
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10. Intranuclear viruses. The naked viruses (V) which measure 108 nm. in diameter have electron dense cores, contain several electron dense particles, or are empty. The enveloped viruses (E) measure 144 nm. in diameter and are located within a nuclear vesicle. 31,920x. 11. Enveloped and naked nuclear viruses within a vesicle. Some of the enveloped viruses are empty and some contain electron dense particles. 51,300x. 12. Two cytoplasmic enveloped viruses that are similar to nuclear enveloped viruses. A nuclear virus is also seen. 39,900x. 13. Enveloped, cytoplasmic viruses (V) measuring 144 nm. in diameter. One virus appears to be budding from a cytoplasmic vacuole (Vac). Cytoplasmic organelles are indistinguishable. One enveloped virus is in the extracellular spaces. 39,9O0x.
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14. Cytoplasmic enveloped viruses. These viruses are similar to nuclear enveloped viruses. 51,300x. 15. The viruses in the extracellular space appear to have budded from the cytoplasmic membrane as is indicated by the projection of the membrane. The viruses have electron dense cores and measure 96 nm. in diameter. 39,900x. 16. Naked viruses (V) being released into the extracellular space as a result of lysis of the cell. Two other structures which resemble disorganized mitochondria (M) are also present. 31.920x. 17. Extracellular viruses closely associated with an intact cytoplasmic membrane. The viruses are naked, have dense cores, and measure 96 nm. in diameter. 39,900x.
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To embed the cells in 100% Epoxy mixture, the pellets were cut into small pieces and placed in the tips of capsules containing a small amount of embedding medium. After the specimens were positioned, capsules were filled and kept at 60° C. for 48 hours for polymerization of the plastic. Ultrathin sections (600 to 700 A. thick) were stained using 2% aqueous uranly acetate (Huxley et al., 1961) for 40 minutes at 60° C. followed by 8 minutes in lead citrate (Reynolds, 1963) at room temperature.
RESULTS AND DISCUSSION In the noninfected cell culture, the presence of herpesvirions was not observed nor was there evidence of any viral infection. The cells were intact, lying adjacent to each other but separated by their cytoplasmic membranes. There was no evidence of cell separation nor degeneration. The nuclei were circular to ovoid and usually occupied a portion of the cell toward one end. The membranes of the nuclei were intact, and the chromatin appeared as a centrally located electron dense mass. The most distinct structures of the cytoplasm were the mitochondria, the rough and smooth endoplasmic reticulum, and masses of polyribosomes. The mitochondria were enclosed by a double layered membrane and contained an organized internal arrangement of cristae. Networks of rough and smooth endoplasmic reticulum were extensive throughout the cytoplasm. In the cell cultures infected with HVT, viral infectivity of ten produced only slight changes in the nuclei and in the cytoplasm; however, in other cells there was complete degeneration with loss of the cytoplasmic and nuclear membranes. In cells in which the membranes were still intact, there was often extensive vacuolation of the cytoplasm which caused distortion of the nuclei. There was limited cytoplasmic space available and marked loss and degeneration of cytoplasmic organelles. There was margination of the nuclear chromatin and usually the presence of several to many herpesvirions could be found scattered in the nuclei and in the cytoplasm. In these cells, the mitochondria appeared to have lost their internal arrangement of cristae and to have decreased in size. The extensive network of rough and smooth endoplasmic reticulum was lost, and only fragments of mostly rough endoplasmic reticulum remained. The presence of aggregates of small nuclear particles was observed in some cells. These particles were found in the presence
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medium 4.5 ml. of the culture fluid were removed from each of the flasks to increase the probability of virus-cell contact and each flask was inoculated with .2 ml. of the HVT virus stock (Merck, Deptavac-HVT). After 3 hours incubation, 4.0 ml. of maintenance medium were added to each flask. Twenty four hours postinoculation the medium was removed from each flask and replaced with new maintenance medium. The cell cultures were examined daily with the light microscope for the development of cytopathic effects (CPE). Four days postinoculation noticeable CPE was observed in the infected flasks and the monolayers were detached from the flasks by a gentle rotation of the flasks. The monolayers from the flasks were pooled and centrifuged in a clinical centrifuge to form a cell pellet. The monolayers from the control flasks were removed with trypsin, pooled and centrifuged. The pellets were suspended in 1% osmium tetroxide prepared according to Millonig (1961) and fixed for 1 hour at 4° C , then dehydrated through graded alcohols and propylene oxide according to traditional manner for epoxy embedment and embedded in an Epon-Araldite mixture composed of two parts of solution A (62 ml. Araldite 506 and 100 ml. dodecenyl succinic anhydride), one part solution B (100 ml. Araldite 506 and 89 ml. nadic methyl anahydride) and 1 / 10th volume Epon 812 with 1.5-2% tris dimethyl aminomethyl phenol-30 as catalyst.
HERPESVIRUS IN FIBROBLASTS
Herpesvirions were observed in all cells in which there were indications of cellular destruction. Most of the virus particles were seen in the nuclei; however, occasionally, viruses were seen in the cytoplasm. In cells containing crystals of nuclear particles, the predominant type of virus was one that contained several electron dense particles which often were arranged so that the center of the virus appeared as an electron lucent cross. This type of virus also occurred in the presence of other types of virions, in the absence of any nuclear particles, and as both a naked and enveloped virus. Other types of naked viruses were those with electron dense cores and those in which the capsid was empty. Naked viruses measured 95 to 110 nm. in diameter.
Enveloped viruses present in the nucleus and cytoplasm were observed only in intact cells. Envelopment apparently occurred by the budding of viruses either into or out of vacuoles. In the nuclei containing both naked and enveloped viruses, the enveloped viruses occurred only within vesicles. In the cytoplasm, the enveloped viruses were seen both free of any enclosing vacuole and budding from a vacuole. Enveloped viruses measured 140 to 160 nm. in diameter. Only naked virions were observed in cells in which karyolysis was complete and in which the cytoplasmic membranes were lost. Naked virions of the three types described previously were observed in the remaining nuclear material; however, viruses in the cytoplasm usually had electron dense cores and were surrounded by granular material. Extracellular viruses associated with cell debris were quite numerous and measured 95 to 100 nm. in diameter, had electron dense cores, were unenveloped, and were usually masked by the deposition of granular material. Viruses were observed in the process of budding both into and out of the cytoplasmic membranes. Viruses budding from the membrane averaged 115 nm. in diameter, had electron dense cores, and were not enveloped. Viruses budding into the cell were unenveloped and averaged 106 nm. in diameter. Viruses were often found closely associated with the cytoplasmic membrane, but were rarely budding. In many cells in which there were few indications of viral infection other than margination of the chromatin, the nuclei contained aggregates of crystals of nuclear particles. The particles measured 35 nm. in diameter, had translucent centers, and were bordered by electron dense material. Aggregates of particles usually occurred at the periphery of the nuclei, and as many as three to four crystals were found in a single nucleus. In nuclei containing the crystals, very few
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of only limited numbers of virions and were located at the periphery of the nuclei. With progression of infection, degenerative effects of the cells were more evident. Gradually, karyolysis occurred and the karyoplasm diffused into the cytoplasm. The nuclear chromatin no longer formed a marginal rim at the periphery of the nucleus but occurred as electron dense clumps intermingled with cytoplasm. Further destruction of mitochondria was characterized by complete loss of internal and external integrity. Other cytoplasmic organelles were lost as the cells progressed to complete disorganization. There was release of naked virions from the nuclear area into the cytoplasm and intercellular spaces due to the focal dissolution of the cytoplasmic membrane. Infection resulted in the formation of polykaryocytes which usually contained two to three nuclei. The absence of distinct cytoplasmic membranes between adjacent cells suggested fusion of the cells resulting in a multinucleated mass of cytoplasm. This characteristic was not observed in control cell cultures. The morphological changes produced by viral infection were similar for polykaryocytes and mononucleated cells.
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The infectivity of the two enveloped viruses has not been determined. Nazerian et al. (1971) suggested that infectivity could be similar to the herpes simplex virions in which the bulk of the infectivity is associated with the cytoplasmic fraction. In a study by Nii et al. (1968) of the sequence of the
development of the group A, herpes simplex virus (HSV), it was concluded that the unenveloped capsid is unstable outside the cell and that the enveloped capsids are the infectious or complete viruses. The mechanism of infectivity of HVT is debatable. There was evidence to suggest that HVT can leave the cell by budding through the cytoplasmic membrane. This was not a common observation and the viruses involved were naked viruses. Extracellular viruses were, however, numerous. Many cells were seen in the process of lysis resulting in the loss of the cytoplasmic membrane with the release of viruses into the extracellular fluid. Aggregates of viruses were found both associated with cell debris far removed from any intact cells and also closely associated with the cytoplasmic membranes of intact cells. The release of viruses from cells did appear to be by rupture of cells rather than by budding from intact cells. With HVT infection, numerous naked viruses were found extracellularly and also budding into cells. This evidence indicates that in vitro HVT does not require the envelope to be infective and that the naked extracellular viruses are mature, functionally infective viruses. REFERENCES Eidson,C.S.,andD. P.Anderson, 1971. Immunization against Marek's disease. Avian Dis. 15: 68-81. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15: 312-322. Epstein, M. A., B. G. Achong, A. E. Churchill and P. M. Biggs, 1968. Structure and development of the herpes-type virus of Marek's disease. U.S. National Cancer Institute J. 41: 805-820. Gravell, M., A. Granoff and R. W. Darlington, 1968. Viruses and renal carcinoma of Rana pipiens, VII propagation of a herpestype frog virus. Virology, 36: 467-475. Huxley, H. E., and G. Zubay, 1961. Preferential staining of nucleic acid-containing structures for electron microscopy. J. Biophysic. Biochem. Cytol. 11: 273-296.
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herpesvirions were present. The viruses observed usually contained either three to four electron dense particles at the periphery of the virion or one centrally located particle. In both types, the enclosed particles were morphologically identical to the nuclear particles. Cells with many herpesvirions did not contain aggregates of the nuclear particles. Viruses in which the capsid was empty might have appeared so due to the particular section cut. Sectioning and the position of the virus could also account for those viruses containing only electron dense particle. As for those viruses having extremely dense cores, it was not possible to determine whether they were primary forms of the virus or perhaps a mature form of those viruses containing several particles, having been formed by the condensation of the particles into one dense mass. Since both forms of the virus have been seen enveloped, this suggestion of maturation seems unlikely. In the nucleus, viruses appeared to acquire the envelope either by budding into nuclear vesicles or by budding through the nuclear membrane into the cytoplasm and the envelope had the appearance of a membrane surrounding the capsid and was somewhat granular. The majority of the nuclear enveloped viruses had electron dense cores. Envelopment in the cytoplasm appeared to occur as viruses budded out of cytoplasmic vacuoles and two types of enveloped were seen. One type was similar to nuclear enveloped viruses and a second type of envelope appeared to be concentrically layered about the capsid which enclosed a moderately electron dense core. The enveloped viruses measured 140 to 160 nm. in diameter.
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Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413-429. Plummer, G., 1967. Comparative virology of the herpes group. Progr. Med. Virol. 9: 302-340. Purchase, H. G., B. R. Burmester and C. H. Cunningham, 1971a. Responses of cell cultures from various avian species to Marek's disease virus and herpesvirus of turkeys. Amer. J. Vet. Res. 32: 1811-1823. Purchase, H. G., and W. Okazaki, 1971. Effect of vaccination with herpesvirus of turkeys (HVT) on horizontal spread of Marek's disease herpesvirus. Avian Dis. 15: 391-397. Purchase, H. G., W. Okazaki and B. R. Burmester, 1971b. Field trials with the herpes virus of turkeys (HVT) strain FC 126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Purchase, H. G., R. L. Witter, W. Okazaki and B. R. Burmester, 1971c. Vaccination against Marek's disease, In: Perspectives in Virology, (Gustav Stern Symposium), edited by M. Pollard, Academic Press, New York, New York, vol. 7, p. 91-110. 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. 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.
NEWS AND NOTES (Continued from page 273) Manufacturing Division, Merck Animal Health Division, and Merck Chemical Division. This reorganization accompanied major changes in executive responsibility and organizational alignment that Merck President John J. Horan said would enhance the company's ability to meet future challenges and opportunities. Dr. Hilmer L. Jones has been elected Vice President and General Manager of the Merck Animal Health Division, formerly the Animal Health and Feed Products area. Dr. Jones had been appointed General Manager of the Animal Health and Feed Products Department in October, 1974, after joining Merck in 1960 as Director of Technical Services for that Depart-
ment. He previously had served in executive positions in the agricultural and veterinary field with Pfizer, Inc. A native of Crenshaw County, Alabama, Dr. Jones received a B.S. degree, majoring in agricultural science, an M.S. degree, majoring in animal nutrition, and a D.V.M. degree at Auburn University. He formerly was Chairman of the Division of Environmental Health, Auburn University Extension Service. He helped organize and develop the Division. He also has been involved in several voluntary activities with veterinary and health planning organizations. In his new position, Dr. Jones reports to John L. Huck, Senior Vice President of Merck & Co., Inc.
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Kaleta, E. F., and R. A. Bankowski, 1972a. Production of circulating and cell-broud interferon in chickens by type 1 and type 2 plaque-producing agents of the Cal-1 strain of Marek's disease herpesvirus and herpesvirus of turkeys. Amer. J. Vet. Res. 33: 567-571. Kaleta, E. F. and R. A. Bankowski, 1972b. Production of interferon by the Cal-1 and turkey herpesvirus strains associated with Marek's disease. Amer. J. Vet. Res. 33: 573-577. Kawamura, H. G., J. King, Jr. and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell cultures of normal turkeys. Avian Dis. 13: 853-862. Melnick, J. L., M. Midulla, I. Wimberly, J. G. Barrera-Oro and B. M. Levy, 1964. A new member of the herpesvirus group isolated from South American marmosets. J. Immunol. 92: 596-601. Millonig, G., 1961. Advantages of a phosphate buffer for O s 0 4 solutions in fixation. J. Appl. Phys. ew: 1637. Mustaffa-Babjee, A., P. Ketterer and P. B. Spradbrow, 1971. Isolation of turkey herpesvirus in cell culture. Aust. Vet. J. 47: 125. Nazerian, K., and R. L. Witter, 1970. Cell-free transmission and in vivo replication of Marek's disease virus. J. Virol. 5: 388-397. 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. Virology, 43: 442-452. Nii, S. Hiro, C. Morgan and H. M. Rose, 1968. Electron microscopy of herpes simplex virus, II Sequence of development. J. Virol. 2: 517-536.
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