Gordon Memorial Lecture. Chicken neoplasia--a model for cancer research.

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Chicken neoplasia—a model for cancer research B. W. Calnek

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Department of Avian and Aquatic Animal Medicine, College of Veterinary Medicine , Cornell University , Ithaca, New York, 14850, USA Published online: 08 Nov 2007.

To cite this article: B. W. Calnek (1992) Chicken neoplasia—a model for cancer research , British Poultry Science, 33:1, 3-16, DOI: 10.1080/00071669208417439 To link to this article: http://dx.doi.org/10.1080/00071669208417439

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British Poultry Science (1992) 33: 3-16

GORDON MEMORIAL LECTURE CHICKEN NEOPLASIA—A MODEL FOR CANCER RESEARCH1 B. W. CALNEK

Downloaded by [University of Montana] at 09:00 03 April 2015

Department of Avian and Aquatic Animal Medicine, College of Veterinary Medicine at Cornell University, Ithaca, New York 14850, USA

Abstract 1. The use of animal models has been immensely important for the advancement of our knowledge of the aetiology and pathogenesis of human diseases, including neoplasia. 2. Viruses, as oncogenic agents, were first described in the early 1900s when cell-free filtrates were used experimentally to transmit leukemias and sarcomas in chickens. In more recent years, studies with avian leukosis/sarcoma viruses have led the field in attempts to establish the genetic and molecular basis of viral oncogenesis. 3. Marek's disease of chickens was the first neoplasm proven to be caused by a herpesvirus and it remains the only neoplastic disease for which an effective vaccine has been developed and deployed. It serves as an elegant model as we seek an understanding of the pathogenesis of herpesvirus-induced lymphomas at both the cellular and molecular levels.

INTRODUCTION

There is a rich history of firsts in oncology which had their roots in studies involving chickens. Truly, much of the current theory and knowledge of the causation of virally-induced cancers in any species, including humans, can be directly traced to observations carried out with poultry. This is not just happenstance; several factors have contributed to the usefulness of avian species as models for cancer research. Perhaps the most significant of these is the prevalence and variety of naturally-occurring neoplasms in chickens, many of which are analogous to those seen in mammalian species. Lymphoid tumours predominate in terms of incidence, with losses in excess of 50% of the flock possible, but a rich spectrum of connective tissue, epithelial, endothelial and other related tumours may also be observed (Payne and Purchase, 1991). The fact that tumours are a major cause of economic loss has spurred research on their causation and prevention for the sake of the host species, complementing the benefits derived in their use as a model for cancer in other species. 1

This lecture, the ninth given in memory of the late Dr R. F. Gordon, was delivered at the School of Pharmacy, University of London, on the 21st of March, 1991.


B. W. CALNEK

B. W, CALNEK—9th Gordon Memorial Lecturer.

Also contributing to the importance of the avian model is the fact that the aetiology of most tumours is known, many are readily transmissible, and the chicken is easily manipulated as a laboratory animal. Development of specificpathogen-free flocks of defined genetic makeup has been essential to the

GORDON MEMORIAL LECTURE

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exploitation of the mod'el, but this was readily achieved in parallel with the understanding and definition of the various aetiological agents involved. This paper can do no more than provide a meagre sampling of the examples of important findings and concepts that have resulted from experimentation with domestic fowl. It will focus on the identification of viruses as oncogenic agents through transmission experiments, the discovery and significance of oncogenes, the importance of pathogenesis studies, and the present and future role of molecular approaches to the understanding of oncogenicity. Examples will be restricted mainly to the leukosis/sarcoma viruses and the Marek's disease herpesvirus. TRANSMISSION OF ONCOGENIC AVIAN RETROVIRUSES AND HERPESVIRUSES

Retroviruses

Leukotic disease in chickens was reported in the 1800s, but it was not until early in the 1900s that any of the many forms was experimentally transmitted. Although others demonstrated the transmissibility of avian tumours slightly earlier, Ellermann and Bang's (1908) report of transmission of erythroblastosis with cell-free material signalled the beginning of the era of viral oncology. This was followed shortly by Dr Peyton Rous' discovery (Rous, 1911) that fibrosarcomas also could be transmitted with tumour filtrates. These contributions preceded by many decades any similar work with other species. They received much less attention than they should have, perhaps because many thought that findings with birds were not particularly relevant to the problem of mammalian cancer. In fact, it was not until 1968 that Rous was recognised for his pioneering work on the Rous sarcoma virus by the award of a Nobel Prize. Not all avian tumours, lymphoid leukosis (LL) for example, were so easily transmitted. This is not surprising now that we understand that not all oncogenic retroviruses carry a fast-acting oncogene which can directly transform cells. In addition, there was the problem that experimental chickens used for transmission were poorly characterised; undoubtedly, many of them were genetically resistant to infection with some of the viruses and others may have carried antibodies to hamper infection. Nonetheless, work continued and genetically susceptible genetic strains free of LL virus eventually were developed. This made it possible to show that not only was LL transmissible using filterable material, but also that it and other viruses were contagious and could be transmitted both horizontally and vertically (Payne and Purchase, 1991). Once the hurdle of effecting experimental transmission was past, progress was rapid. In a choice bit of serendipity while working on Rous sarcoma virus (RSV) in cell cultures, Rubin (1960) discovered that avian leukosis virus (ALV) conferred resistance to RSV. This led to his famous resistance-inducing factor (RIF) test for ALV which, in turn, permitted further definition of the epidemiology, pathogenesis and immunology of lymphoid leukosis. Although studies on the various manifestations of infection by leukosis/sarcoma viruses has led to an understanding of the multipotent nature of this agent group, the types of

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target cells involved and the characteristics of neoplastically transformed cells, the most rewarding harvests from the avian model have derived from studies on the viruses themselves, their genes, and their interrelationship with host cell genes (see section on oncogenes). Unquestionably, the most important virus of all was the Rous sarcoma virus. Had it not been discovered through transmission attempts, viral oncology would have most certainly followed a much more difficult pathway.


Herpesviruses

Marek's disease, another lymphoma, was particularly difficult to transmit; attempts by a number of research groups gave equivocal results at best. This was discouraging in the face of evidence that the disease spread easily within a flock. In this case, the breakthrough in transmission also depended on the help of geneticists who developed flocks with high susceptibility. Independently, and nearly simultaneously, Sevoian et al. (1962) and Biggs and Payne (1963) developed models for studying the disease using cellular inocula and genetically susceptible strains of chickens. The importance of the highly susceptible Sstrain for transmission and subsequent work on MD cannot be overemphasised. Developed almost as an "example of contrast" during the classic research carried out by Hutt and Cole (1947), this strain turned out to have far greater impact than the genetically resistant K and C strains which formed the objective of their work. With Marek's disease (MD), experimental transmission soon led to the isolation of the causative agent, a highly cell-associated herpesvirus (Churchill and Biggs, 1967; Nazerian et al., 1968; Solomon et al., 1968). The importance of MD as a model became apparent soon thereafter when cell-free virus was isolated from the feather follicle epithelium, permitting for the first time the reproduction of the disease using virus alone (Calnek et al., 1970). This was the first definitive proof that a herpesvirus could cause a neoplastic disease; similar transmission studies were impossible with the Epstein-Barr virus which had been linked to Burkitt's lymphoma only by circumstantial evidence. Indeed, there are few animal models for the several human herpesviruses known or suspected to be oncogenic. None present the advantages of Marek's disease in which transmission of the virus to the natural host results in a highly reliable reproduction of the disease. That is why Marek's disease is the best characterised of all oncogenic herpesviruses in terms of influential factors such as virus pathogenicity, genetic susceptibility, immune response, etc. In addition, the in vivo studies thus permitted were essential for the identification of attenuated and nononcogenic strains of virus. These in turn formed the basis for the monumentally significant development of vaccines against the disease. On the other hand, the use of the MD model for identification of genes important to neoplastic transformation is only now being exploited (see below). DISCOVERY AND SIGNIFICANCE OF ONCOGENES

Klein (1988) stated that "The development of this field came about



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through a series of historical accidents . . . the first oncogenes were discovered as a byproduct of the research on RNA tumour viruses". Avian tumour viruses were central to the entire issue, especially the leukosis/sarcoma group. In 1960, Rubin accidentally discovered that a factor associated with cells from some chicken embryos could induce resistance to the acutely-transforming Rous sarcoma virus (RSV); this "resistance-inducing factor" (RIF) was shown to be none other than our well known avian leukosis virus. Beyond that point, however, wisdom linked to hindsight seems to suggest a sequence of important and immensely rewarding studies that led in a logical, rather than accidental, way to the present understanding of oncogenes and their role in cancer. Rubin's findings of RIF pointed the way for Hanafusa's discovery of defectiveness of some strains of sarcoma virus (Hanafusa et al., 1964) which contained apparently transforming genes but could not replicate without the help of complementary genes from "helper" viruses. The transforming gene, later identified as "src", proved crucial to our understanding of oncogenes. In 1964, Temin made the heretical proposal that during RSV replication, a "provirus" is produced which is composed of DNA, although the virus itself is made of RNA. This implied that if the provirus is composed of DNA, then the life cycle must involve the transfer of information from the viral RNA to DNA of the provirus and thence to the RNA of progeny virus. He proved the point in experiments using inhibitors of DNA synthesis (BUDR) or DNA-directed RNA synthesis (actinomycin D) in which he showed the need for an early but not late period of DNA synthesis, and the need for DNA-directed RNA synthesis thereafter for the production of infectious virus. Indeed, he was able to show homology between labelled RNA from RSV and DNA from RSVinfected cells. Finding viral genes integrated as DNA in the host cells, while not directly contributory to the discovery of oncogenes, remains a critical point in our understanding of the relationship between viruses and oncogenes. The discovery of the molecular mechanism for the antidogmatic conversion of RNA to DNA, rather than the reverse, also depended on the avian virus model in large part; Temin (Temin and Mizutani, 1970) and Baltimore (1970) simultaneously reported on the finding of reverse transcriptase in virions of RSV in 1970. They received Nobel Prizes for their discovery shortly afterward; clearly the chicken virus model was gaining acceptance as a worthy subject! More direct impact on the development of the oncogene theory which was to be proposed by Huebner and Todaro in 1969 was provided by Dougherty and Di Stefano (1966) and Dougherty et al. (1967). They examined chicken embryos from a number of breeding flocks, including several maintained in isolation and free of avian leukosis virus infection, and found a surprisingly high proportion (over 50%) had typical C-type virus particles in the pancreas. However, no infectious virus could be demonstrated. From 50 to 90% of embryos from different flocks had leukosis virus group-specific (gs) antigen detectable by a complement-fixation test (COFAL) but there was no correlation between that and the leukosis virus infection status of the individual embryos. They concluded that the leukosis virus gs antigen was similar or identical to a normal chicken antigen. Payne and Chubb (1968) confirmed those results and


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showed that the gene(s) involved in production of gs antigen were inherited in a simple Mendelian fashion. Prophetically, they noted that the avian leukosis virus could have acquired part of its genome from an avian host, perhaps by a process analogous to transduction in the phage-bacterium interaction. Subsequent studies (Hanafusa and Hanafusa, 1968; Weiss, 1969) identified another endogenous viral gene product, chf, which constituted envelope glycoproteins. At this point, the stage was set for the oncogene theory. Huebner and Todaro (1969) proposed ". . . that there exists a unique class of viruses present in most, and perhaps all, vertebrates that plays an important etiologic role in the development of tumours in these animals". They believed that viral information was passed vertically to progeny animals or cells as a repressed viral genome, and they predicted that all cancers are the result of expression of the oncogenic information (the oncogene) of covert C-type RNA viruses. Much of this was based on the knowledge that avian and mouse viruses could be readily transmitted. The genetic and antigenic data provided from the avian model on the presence of cellular genes which could code for an avian leukosis viral group-specific antigen weighed heavily in their theory. They could not know that their putative oncogene(s) was, in fact, of cellular rather than viral origin and that some retroviruses apparently acquired cellular genes which could be thereafter modified through mutation or other means to become highly oncogenic. Klein (1988) characterised the event as ". . . accidental illegitimate recombination, facilitated by the retroviral lifestyle that consists of a perpetual series of two-way transitions between the freely movable viral RNA stage and the integrated proviral DNA". Currently, there are over 40 known oncogenes which are similar in all vertebrates and can be grouped into categories such as growth factors, growth factor receptors, protein kinases, signal transducers, nuclear oncogenes and transcription factors; all are involved in the regulation of cell division (Klein, 1988). The avian virus models again were important contributors to our knowledge. Significantly, the first identified oncogene was src, found during genetic and biochemical analyses of avian RSV by Duesberg, Vogt, Martin, Stehelin and others in the early 1970s (Bistner and Jansen, 1986). It is a protein kinase whose product, a 60,000 dalton phosphoprotein, is associated with the plasma membrane. Its function is still unknown, but it could act by alteration of a membrane receptor. Myc, from MC29 (an acute leukemia virus of chickens), is an example of a nuclear oncogene. It codes for a DNA-binding nuclear phosphoprotein that can transactivate other genes involved in cell proliferation. The switch-on of cellular myc by an adjacent translocation of an immunoglobulin locus in Epstein-Barr virus-infected B lymphocytes apparently prevents terminal differentiation and thus the cells continue to proliferate, leading to Burkitt's lymphoma. It also appears to be the relevant cellular oncogene in avian leukosis; ALV long terminal repeats (LTRs) serve as a potent promoter when inserted next to c-myc in bursacytes. This widespread, highly conserved oncogene is now thought to be involved in both viral and nonviral human malignancies (Bistner and Jansen, 1986).


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From all of this it is clear that the chicken and its tumour viruses have played an enormously important role in shaping and directing the research which resulted in our present understanding of viral oncology, viral and cellular oncogenes, and their significance in cancer induction.


HERPESVIRUSES, CANCER AND MAREK'S DISEASE

That herpesviruses can cause cancer is undisputed. There are few vertebrate species that do not have at least one herpesvirus to which they are susceptible. Luckily, although the potential is probably there, most herpesviruses do not have neoplasia on their agenda; they generally are content to wreak havoc through necrotising infections. Why and how certain herpesviruses express oncogenic potential are important and intriguing questions. Of the 7 human herpesviruses described to date, the Epstein-Barr virus (EBV) has been best characterised in terms of oncogenicity. EBV infection in humans is extremely widespread and it is most significant as a cause of infectious mononucleosis. In addition, it is the primary cause of Burkitt's lymphoma, a neoplasm of B-cells, and nasopharyngeal carcinoma, and therein lies the stimulus for much of the research that has been carried out on EBV infection. Other oncogenic herpesviruses are associated with tumours in frogs, rabbits, subhuman primates and, of course, chickens. Unfortunately, the study of oncogenic herpesviruses has not been easy. There is no good animal system for EBV, and many of the so-called oncogenic herpesviruses for which there is an animal model are rarely oncogenic in the natural host. Herpesviruses from simians such as H. saimiri, H. ateles and H. papio are good examples of the latter point. EBV studies have been mainly restricted to in vitro transformation models, and those with other viruses must be carried out in an abnormal host or with poorly characterised animal populations. Marek's disease in the chicken has been the exception. As anyone in the poultry industry can attest, MD is anything but a rare event in commercial chickens, the natural hosts. Indeed, very high security isolation is required to keep the infection out of specific-pathogen-free flocks. Furthermore, because of the development of genetically-susceptible strains of chickens, regular and dependable transmission of infection leading to tumour formation has been possible. That, in turn, has permitted detailed study of the pathogenesis of infection and the determination of virus and host factors that influence the likelihood that infection will culminate in lymphoma development. No other system provides such an opportunity. Practical questions dominate early studies on an infectious disease. What is the aetiology? How is the agent transmitted? What is the pathogenesis of infection? What factors influence the severity of disease? What type(s) of immunity develops, and against what? How can the disease be controlled? Is eradication possible? Are vaccines feasible? It is after most of these questions are answered that we have the luxury of truly exploiting an animal model for the advancement of basic knowledge about the mechanism of disease. In the case of oncogenic herpesvirus infections, some of the definitive questions that arise are: What is the nature of the malignancy itself? Is it a true neoplasm?


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What makes the virus oncogenic, that is, how is it distinguished from similar but non-oncogenic viruses? Does oncogenicity derive directly from the virus genome through a viral oncogene, or is the effect indirect through disruption of cellular gene regulation? Marek's disease research evolved very much along these lines. Undoubtedly, it has enjoyed a great advantage over other potential models of herpesvirus oncology in that the disease was, and continues to be, a serious economic threat to poultry production. This led many laboratories into research to answer the practical questions, but at the same time it was impossible to ignore the intriguing complexities of the disease; the scientific potential was so great that many other laboratories interested only in basic aspects of oncogenic virology felt compelled to join in the excitement. Thus, the Marek's disease model continues to be significant long after the original objectives of practical control of the disease were addressed and met. What does the model contribute? This is best answered by a brief review of key findings over the years and a preview of what is yet to come. Importance of the host genotype

There is no better example than Marek's disease of the importance of choosing the right parents to reduce the threat of cancer. Hutt and Cole, in classic studies beginning in the 1930s (Hutt and Cole, 1947), proved the heritability of resistance by developing strains of chickens able to withstand natural exposure levels lethal for a parallel susceptible strain. Some years later, it could be shown that the resistance was not against infection per se but rather against the progression of infection to tumour development (Fabricant et al., 1977). This was highly instructional because it suggested that it is the host response to infection that is crucial. But what is the mechanism? Clues came from studies showing that genetic resistance is obviated following imposed immunosuppression (Sharma et al., 1977) and that MD vaccines work best in genetically-resistant chickens (Spencer et al., 1974). Thus, one might assume that the most responsive chickens would be the most resistant to MD. Genes within the major histocompatibility complex, especially those associated with the B alleles, were found important and at least one major type of genetic resistance was proven to be related to specific B alleles, for example B2i. It is probably correct to deduce that a good immune response is fundamental to genetic resistance, but responsiveness may be a two-edged sword. A number of studies showed that certain measures of cell-mediated immune (CMI) responsiveness were greater in susceptible strains than in their resistant counterparts (see Calnek, 1985). This helped lead to a hypothesis that has important implications regarding the pathogenesis of the neoplastic response to MDV infection (see below). Regardless, the discovery that resistance to a neoplastic disease could be modulated by genetic selection was an important step in our understanding of oncogenicity. Also significant was the observation that genetic resistance is only relative and depends on the virulence of the infecting virus. Thus, even the most susceptible strains of chickens may have a low incidence of neoplasms after infection with a low-virulence isolate of virus,


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whereas other isolates are so virulent that they may cause a high tumour incidence even in the most resistant strains (Schat et al., 1981). Again, an interaction with immune competence can be shown because immunosuppression enhances the tumour incidence with relatively low-virulence viruses (Calnek etal., 1977). Pathogenesis There is no herpesvirus infection that has been dissected so thoroughly as that causing Marek's disease. The sequential events are well established (Calnek and Witter, 1991): during the first 2 to 3 d, infection is carried from the point of entry (respiratory tract) to target cells in the primary lymphoid organs (spleen, thymus, bursa of Fabricius). There, the major target cells are B lymphocytes. Severe cytolytic infection ensues, with an intense inflammatory reacion as the consequence. Activated T cells, stimulated by the necrotising infection, also become cytolytically infected. After 6 or 7 d, humoral and cellmediated immune responses develop and, coincidentally, the infection of both B and T cells enters latency. It is at this point that genetic constitution, virus virulence, and age all enter the picture. For resistant birds which are old enough to be immunologically responsive, and are infected with strains of virus other than those with exceptional virulence, the story is essentially complete. A low-level, persistent latent infection (mostly T lymphocytes) continues for the life of the bird with no further obvious effects. However, for the genetically-susceptible bird, or one infected with a very highly virulent strain of virus, or for a bird which is immunosuppressed, a second series of events occurs beginning at 2 to 3 or more weeks after infection: cytolytic infection of lymphoid organs and epithelial-origin tissues emerges to cause focal areas of necrosis and inflammation; a permanent immunosuppression occurs, and transformed activated T cells proliferate at a variety of sites resulting in lymphoma formation. Nonneoplastic inflammatory lesions may develop. These are especially evident in nervous tissue, but tumours are the prominent end lesion. Several observations helped shape continued studies on pathogenesis. First was the finding that genetically-susceptible chickens often had superior CMI responses to mitogenic stimulation (see Calnek, 1985). This did not fit the obvious conclusion that resistance should be associated with a good immune response. Secondly, it was learned from studies with MD tumour cell lines that, whereas B cells are a primary target for cytolytic infection with MDV, T cells are the targets for transformation; virtually all of the many lines developed were of T cell origin. Third came the discovery that T cells are only susceptible to infection if they first become activated, as they would in a conventional immune response (Calnek et al., 1985). These points directed us to the hypothesis that susceptibility to MD is correlated with a superior T-cell response against antigens seen in the early cytolytic infection of B cells. This would provide an abundant supply of activated T-cell targets for transformation (Calnek, 1986). In searching for a way to test the hypothesis, we reasoned that enhanced


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stimulation of T-cell activation would lead to enhanced tumour induction. The development of a local lesion model, in which alloantigens were injected together with MDV in the wing web or pectoral muscle, proved the point; tumours developed at the site of inoculation (Calnek et al., 1988; Calnek etal., 1989). Perhaps the most important contribution of the local lesion model was the coincidental finding that cells from the lesion site could be used to initiate MD tumour cell lines even when harvested as early as 4 d after inoculation. This meant that either cells had already become transformed during the early cytolytic phase of infection, or they had transformed in vitro. Scores of cell lines derived using the local lesion model have offered new insights into the pathogenesis of MD. For instance, contrary to earlier beliefs that a specific subset of T cells was probably involved as tumour cells, we can now say that probably any T-cell subset is susceptible to transformation. Collaborative studies using a battery of monoclonal antibodies against surface markers showed that cell lines may be CD4+ (helper T cells), CD8+ (cytotoxic/suppressor T cells), or may have neither marker (unknown function), and either TCR2 or TCR3 receptors may be expressed on each of these subsets (Schat et al., 1988; Schat et al., 1991). Thus, the deciding factor seems to be more a question of what cell type(s) predominates at a site of virus infection rather than a predilection of a certain type to transformation. Also, we have learned that genome expression varies greatly among cell lines, even those of the same phenotype, because the proportion of cells with viral antigen can range from a fraction of 1% to as high as 30 to 40%. This suggests variable intrinsic control mechanisms. The inclusion of cytokines associated with "conditioned medium" in the medium of developing cell lines can inhibit the production of viral antigens pointing out that extrinsic factors are also important. The model now must be exploited to determine what transcripts are essential for induction and maintenance of the transformed state, and if transformation is under viral or cellular control, or both. The local lesion model encouraged attempts to transform cells following in vitro infection of activated T cells. Three promising lines were developed (Calnek and Schat, 1991), but apparent inability of the cells to survive beyond 100 to 150 culture d begs the question of whether or not they were truly transformed. That, in turn, raises the issue of exactly what constitutes a marker for transformation, because cell lines could be immunologically driven or caused to proliferate without concomitant immortalisation. Vaccines

No discussion of Marek's disease as a model for cancer research would be complete without mention of the enormously successful conclusion of the first major chapter of MD research. One must remember that at the beginning of the 1960s, it was not possible to transmit MD, the aetiology was a complete mystery, and the only hope for control of this devastating disease rested on the shoulders of poultry geneticists. The transmission studies reported by Biggs and Payne (1963) and Sevoian et al. (1962) noted above opened the floodgates and allowed, for the first time, characterisation of the agent. Its cell-associated



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nature was soon described and a race to discover the identity of the agent ensued. Cell culture isolation attempts were successful in two laboratories; the 1967 description of a herpesvirus etiology by Churchill and Biggs (1967) in England was soon followed by a similar report from Nazerian and coworkers in the United States (Nazerian et al., 1968; Solomon et al., 1968). Churchill and coworkers at Houghton, England soon thereafter successfully attenuated the virus (Churchill et al., 1969a) and showed that it was not only no longer oncogenic, but protected chickens against challenge with virulent MD (Churchill et al., 19696). Commercial vaccine was soon available, constituting the very first application of a cancer vaccine in any species. The breakthrough had been made, although the implications went largely unnoticed outside the poultry industry. There remained only the refinements of the approach and the development of other vaccines. Workers at East Lansing in the USA followed with the finding that a naturally non-oncogenic turkey herpesvirus (Kawamura et al., 1969; Witter et al., 1970) was also protective (Okazaki et al., 1970). Other vaccines consisting of very low virulence MDV from chickens (Rispens et al., 1972) or naturally non-oncogenic chicken herpesviruses (Schat and Calnek, 1978; Zander et al., 1972) rounded out a battery of vaccines that have been used worldwide for two decades with a very impressive record of safety and efficacy. Over 90% protection can be expected under commercial conditions. Unfortunately, under the pressure of vaccination, more virulent strains of MDV have emerged, requiring newer strategies such as the use of combinations of vaccines (Calnek et al., 1983; Witter et al., 1984). These so-called bivalent or polyvalent vacines, generally including the turkey herpesvirus and a serotype 2 non-oncogenic chicken herpesvirus such as SB-1, apparently offer the benefit of synergism between the components (Witter, 1988). To date, the MD model of vaccination to control a neoplastic disease has not been applied to any of the oncogenic herpesvirus infections in other species, although other herpesvirus vaccines are now being used, including an attenuated human varicella-zoster vaccine. It seems quite clear that protection is immunologically based because effective vaccination requires immunological competence. Vaccination is probably directed against early antigens; it interrupts the usual pathogenesis of infection by preventing the early cytolytic infection (Smith and Calnek, 1974). Perhaps its main effect is to preclude the generation of large numbers of activated T cells important to the earlier stated hypothesis. Molecular studies

The Marek's disease model must now assume an important role in the establishing a molecular basis to transformation by a herpesvirus. Molecular biology has been a difficult area of research with MD, largely because the virus is very cell-associated. So far, molecular studies on MDV have concentrated on determining the physical properties, structure, and transcription of the viral DNA, and on mapping of viral genes (Ross, 1985; Hirai, 1988). Some differences have been observed between oncogenic and non-oncogenic strains or


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between oncogenic strains of different levels of virulence, but no solid clues as to the basis to the oncogenicity have yet been identified. Even so, the model has a number of advantages which demand continued study and exploitation. The system is the more remarkable because of the qualitative and quantitative differences in oncogenicity among serotypes and strains of virus. Two of the three known serotypes are considered to be non-oncogenic, while the third has virus strains that vary from very weakly to very strongly oncogenic. Furthermore, attenuated variants for the oncogenic serotype-1 viruses can be developed. These reagents are ideal for comparative viral genetic studies, even though the cell-associated character of the viruses has made methods difficult. Both in vivo and in vitro systems for the propagation and study of replication and transformation are now in place. The local lesion model described above probably represents a system in which cells become infected in vivo but transform in vitro. This permits studies on the relative importance of host genotype, T cell subset and viral strain away from host influences. The marvellous array of MD tumour cell lines now available (approaching 200 in our collection at Cornell) offers abundant material for the study of gene transcription, integration, translocation and transactivation. Genomic libraries of the various virus types have become, or are rapidly becoming, available. When coupled with developing technology for transfection of avian lymphocytes, they offer the opportunity to identify both immunogenic and transforming genes. For instance, transfection of selected viral genes into potentially transformable target cells could be used in attempts to demonstrate transformation. Also, co-transfection of viral DNA from non-oncogenic or attenuated virus strains plus selected DNA fragments from an oncogenic strain could result in oncogenic recombinants, thus pinpointing the location of genes conferring oncogenicity. Clearly, the model of Marek's disease has much to offer and is likely to provide yet more insights into the mechanism(s) by which a herpesvirus might cause neoplastic disease.

REFERENCES BALTIMORE, D. (1970) Viral RNA-dependent DNA polymerase. Nature, 226: 1209-1211. BIGGS, P.M. & PAYNE, L J . (1963) Transmission experiments with Marek's disease (fowl paralysis). Veterinary Record, 75: 177-179. BISTNER, K. & JANSEN, H.W. (1986) Oncogenes in retroviruses and cells: Biochemistry and molecular genetics. Advances in Cancer Research, 47: 99-188. CALNEK, B.W. (1985) Genetic Resistance, in: PAYNE, L.N. (Ed.) Marek's Disease, pp. 293-328 (Martinus Nijhoff, Boston).' CALNEK, B.W. (1986) Marek's disease—a model for herpesvirus oncology. CRC Critical Review Microbiology, 12: 293-320. CALNEK, B.W. & SCHAT, K.A. (1992) Proliferation of chicken lymphoblastoid cells after in vitro infection with Marek's disease virus. Avian Diseases, in press. CALNEK, B.W. & WITTER, R.L. (1991) Marek's Disease, in: CALNEK, B.W., BARNES, H.J., BEARD,

C.W., REID, W.M. & YODER, H.W. (Eds) Diseases of Poultry, 9th ed., pp. 342-385 (Iowa State University Press, Ames, Iowa).


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CALNEK, B.W., ADLDINGER, H.K. & KAHN, D.E. (1970) Feather follicle epithelium: a source of enveloped and infectious cell-free herpesvirus from Marek's disease. Avian Diseases, 14: 219-233. CALNEK, B.W., FABRICANT, J., SCHAT, K.A. & MURTHY, K.K. (1977) Pathogenicity of low-virulence

Marek's disease viruses in normal versus immunologically compromised chickens. Avian Diseases, 21: 346-358. CALNEK, B.W., SCHAT, K.A., PECKHAM, M.C. & FABRICANT, J. (1983) Research note—field trials

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Gordon Richards Memorial Lecture.

The Gordon Wilson Memorial Lecture.

THE GORDON WILSON MEMORIAL LECTURE.

THE GORDON WILSON MEMORIAL LECTURE.

Gordon memorial lecture. Physics and physiology of incubation.

Gordon Bell memorial lecture. Management of soft tissue sarcoma.

Gordon memorial lecture. Vaccines and vaccination--past, present and future.

Memorial. Edgar Stillwell Gordon.

The Gordon-Taylor memorial lecture: metatropoi aurai--the winds of change.

Masahiro Iio Memorial Lecture.

THE GORDON WILSON LECTURE EVOLUTION OF CLINICAL CANCER GENETICS.

The Gordon Wilson Lecture. The new genetics.

The Gordon Wilson Lecture. Congenital adrenal hyperplasia.

Gordon-Taylor Lecture, 1976. Sir Gordon Gordon-Taylor, surgeon-anatomist and humanist.

The Gillies Memorial Lecture 1975.

Karnofsky Memorial Lecture. The immunotherapy and gene therapy of cancer.

Gordon Wilson Lecture: Infectious Disease Causes of Cancer: Opportunities for Prevention and Treatment.

Hayes Martin Memorial Lecture. Non desperandum.

Second annual Ludwig von Bertalanffy Memorial Lecture.

First Tigani El Mahi Memorial Lecture.

The Cairns memorial lecture: the Cairns tradition.

Phillip Gett memorial lecture awareness in anaesthesia.

Edward B.D. Neuhauser memorial lecture. Bronchopulmonary dysplasia and research in diagnostic radiology.

The 41st David A. Karnofsky Memorial Award Lecture: Academic research worldwide--quo vadis?