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Immunology of Oncornaviruses
Symposium on Allergy in Small Animal Practice
Immunology of Oncornaviruses
William D. Hardy, Jr., V.M.D.*
Virtually all organisms can be parasitized by viruses: vertebrate and invertebrate animals, plants, fungi, and bacteria. Some viruses are ubiquitous but are not known to cause a specific disease while other viruses cause diseases ranging from mild to fatal syndromes. Viruses which cause cancer are termed "oncogenic viruses." Both RNA and DNA viruses are etiologic agents of naturally occurring neoplasms in various animal species (Table 1). RNA viruses that cause naturally occurring neoplasms are found in chickens, cats, hamsters, mice, and primates. DNA viruses that cause naturally occurring neoplasms are found in frogs, chickens, rabbits, squirrels, dogs, cows, and even in man. The most significant oncogenic DNA viruses are the herpesviruses which cause leukemia in primates (Herpesvirus saimiri), renal adenocarcinomas in frogs, and neural lymphomatosis (Marek's disease) in chickens. The first oncogenic virus vaccine has recently been developed against the Marek's disease virus of chickens. This article will deal with the oncogenic RNA viruses and will specifically discuss the immunology of the feline oncogenic RNA viruses as important examples of these viruses in veterinary medicine. The feline leukemia and sarcoma viruses cause naturally occurring cancer in pet cats. 9 The feline sarcoma virus (FeSV) is an immunologically indistinguishable mutant of the feline leukemia virus (FeLV). FeSV causes malignant transformation of fibroblasts while FeLV causes malignant transformation of lymphoid cells (Table 2).12 FeL V is also associated with, and is thought to be the etiologic agent of, myeloproliferative disorders and nonregenerative anemias of cats.7 The cat has the highest incidence of lymphoreticular malignancies of any animal. Lymphosarcoma accounts for 90 per cent of all feline
*Associate,
Head; Laboratory of Veterinary Oncology, Memorial-Sloan Kettering Cancer Center, New York, New York; Consultant in Oncology, Henry Bergh Memorial Hospital of the American Society for Prevention of Cruelty to Animals and The Animal Medical Center, New York, New York.
Veterinary Clinics of NOlth America- Vol. 4, No. I, February 1974
133
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WILLIAM
Table 1.
D.
HARDY, JR .
Oncogenic Viruses Causing Naturally Occurring Neoplasms DNA VIRUSES
RNA VIRUSES
Oncornaviruses Avian leukosis virus Avian myeloblastosis virus Avian erythroblastosis virus Avian sarcoma virus Avian reticuloendotheliosis virus Feline leukemia virus Feline sarcoma virus Hamster leukemia virus Murine leukemia virus (Gross) Murine mammary tumor virus Murine osteosarcoma virus Simian sarcoma virus (Wolly monkey)
Herpesviruses Herpesvirus saimiri (primate) Lucke tumor virus (frogs-renal adenocarcinoma) Marek's disease virus (neural lymphomatosis of chickens) Papovaviruses Bovine papilloma virus Canine oral papilloma virus Deer fibroma virus Human wart virus Rabbit oral papilloma virus Shope papilloma virus Poxviruses Hare fibroma virus Rabbit fibroma virus Rabbit myxoma virus Squirrel fibroma virus Yaba tumor virus
hematopoietic neoplasms. The annual incidence of lymphosarcoma is 41.6 cases per 100,000 cats in the population at risk, and represents onethird of all feline neoplasms.2 Thus, the FeL V is a very important virus in clinical veterinary medicine.
ONCORNA VIRUSES There are marked similarities among oncogenic RN A viruses which distinguish them from other RNA viruses. Previous designations for these viruses were "Ieukoviruses," "B-type and C-type particles," and Thylaxoviridae (sac-like viruses). However, none of these related to the most outstanding property of these viruses, namely, their ability to induce malignant transformation in the cells they infect. Therefore, the term "oncornavirus" (onco, "oncogenic" + rna, "ribonucleic acid") seems the most appropriate term to apply to this class of oncogenic RNA viruses.1o Table 3 lists the known oncornaviruses with their species of origin and notes if their oncogenicity has been proven. Oncornaviruses are widespread in the vertebrate phylum from reptiles to primates. Oncornaviruses are extremely important in veterinary medicine since the avian and feline oncornaviruses are the etiologic agents of the majority of cancer cases in these species. They have been found in association with bovine lymphosarcoma but their oncogenic potential still remains to be elucidated. Recently, oncornaviruses have been
.... ~ ~
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oz
Diseases Associated with Feline Oncornaviruses
C"l
o :
Feline Leukemia Virus
> ~Mutant
Feline Sarcoma Virus
<:
;; c:VJ t'1
VJ
1
Fibroblast Erythroid cell
Lymphoreticular cell
1
Lymphosarcoma or Reticulum cell sarcoma
~
Nonregenerative anemias
I
Myeloid cell ~
Erythroleukemia or Erythremic myelosis
1
1
Fibrosarcoma
Reticuloendotheliosis or Myelogenous leukemia
....
~
(J1
136
WILLIAM
Table 3.
D. HARDY, JR.
Occurrence of Oncornaviruses in Animals ONCOGENICITY OF
ANIMAL
ONCORNAVIRUS
ONCORNAVIRUS
FOUND
PROVEN
Reptiles Snake
+
Avian Chicken
+
+
Mammals Rodents Mouse Rat Guinea pig Hamster
+ + + +
+
Cow Pig Cat
+ + +
Lower primates Rhesus monkey Wolly monkey Gibbon ape
+ + +
Man
+
+ +
+
found in the Rhesus monkey, Wolly monkey, and Gibbon ape. The oncogenic capabilities of the Wolly monkey sarcoma virus have been demonstrated and it only appears a matter of time until human oncornaviruses are discovered.
BIOCHEMICAL PROPERTIES OF ONCORNAVIRUSES
I. All oncornaviruses have a 60-70S RNA. These fast sedimenting RNA's can be converted to 36-37S components by heating or by treatment with dimethylsulfoxide. Such a large viral RNA strand and the conversion of an RNA to a lower sedimentation coefficient are not characteristic of non-oncogenic RNA viruses. 2. In their native state the oncornaviruses do not have hemagglutinating activity. 3. In contrast to non-oncogenic RNA viruses, the production of oncornaviruses can be inhibited, in vitro, by actinomycin D. Since actinomycin D inhibits DNA synthesis, it was hard to understand why this drug should inhibit the production of RNA oncogenic viruses. Within the last 2 years this paradox has been explained by the monumental discovery that oncornaviruses initiate DNA synthesis specified by their viral RNA. The re-
IMMUNOLOGY OF ONCORNAVIRUSES
137
. action is mediated by an oncornavirus reverse transcriptase enzyme (RNA dependent DNA polymerase). This discovery has modified the central dogma of molecular biology which stated that DNA directed RN A synthesis. 4 . Oncornaviruses possess an enzyme, RNA dependent DNA polymerase, that produces DNA from viral RNA template. This enzyme enables oncornaviruses to insert a DNA code, specified by viral RNA templates, into the cellular chromosomes (cellular genome) . This new information may then be passed to subsequent cell generations. In the case of germinal cells, passage of viral-specified DNA to the next generation may result in vertical transmission of viral information.
BIOLOGICAL PROPERTIES OF ONCORNAVIRUSES
1. Oncornaviruses are usually oncogenic in their natural hosts. Three categories of neoplasms are induced by oncornaviruses: (1) leukemias, (2) sarcomas, and (3) mammary tumors (mouse and Rhesus monkey). These viruses may also cause other types of neoplasms as well as non-neoplastic diseases. 2. Oncornaviruses are widespread throughout the vertebrate phylum from reptiles to primates. 3 . Some oncornaviruses are transmitted chiefly vertically (murine oncornaviruses), while others are transmitted chiefly horizontally (feline oncornaviruses) . Vertical transmission occurs when oncornavirus RNA specifies DNA (virogene) which is then integrated into the chromosomes. Oncornaviruses can be activated after birth by activating the virogene (DNA) which produces viral RNA and viral particles. Until recently, it was thought that most oncornaviruses were transmitted exclusively vertically, but our findings of horizontal transmission of FeLV in cats have altered this concept. I, 8 4. Oncornaviruses are not cytopathic. This characteristic allows permanently infected virus-producing cell lines to be established. In animals, these viruses do not kill the cells in which they replicate. The neoplastically transformed infected cells continue to multiply, thus producing tumors.
CLASSES OF ONCORNA VIRUS ANTIGENS Three classes of antigens are associated with oncornaviruses and the cells they infect (Fig. 1 and Table 4). These antigens are (1) virion antigens, (2) cell surface antigens, and (3) soluble antigens.
Virion Antigens There are two types of virion antigens: (1) envelope (type-specific) lipoprotein antigens which are derived from the infected cell mem-
138
WILLIAM
D.
HARDY, JR.
INFECTED CELL VIRION ANTIGENS
Envelope antigens
o
~
.______@'--I 1.......' ..._ (group-specific)
Viral envelope } CELL SURFACE ANTIGENS
.
..............Viral sPf!C!fied ~ non-VIrion
·:.A~:\}:~luble antigens Figure 1.
Antigens associated with oncornaviruses.
brane, and (2) internal (group-specific) antigens located inside the VIrIon.
Envelope antigens are demonstrable by neutralization of infectivity in vitro or in vivo by complement fixation, immunodiffusion, viral interference tests and by immuno-electromicroscopy with visually labeled antibody (hybrid antibody). The demonstration of internal virion or group-specific (gs) antigens by immunodiffusion, complement fixation, and the paired radioiodine labeled antibody tests requires disruption of the viral envelope. Detection of gs antigens in infected cells requires destruction of the cell membrane by acetone fixation to allow penetration of specific antibody into the cytoplasm, the site of synthesis of these antigens. Cell Surface Antigens Cell surface antigens of oncornavirus infected cells are: (1) viral envelope components incorporated in the plasma membrane during the process of viral maturation, and (2) viral-specified cell surface components which are nonstructural elements of the virion. The former cell surface antigens are detectable by immuno-electronmicroscopy. Viral specified non virion cell surface antigens are analogous to the tumor-
139
IMMUNOLOGY OF ONCORNAVIRUSES
specific transplantation antigens (TST A) specified by DNA oncogenic viruses. These antigens are demonstrable by immunofluorescence, immuno-electromicroscopy, and the cytotoxic test.
Soluble Antigens Soluble antigens are the third class of antigens and pervade the body fluids of animals infected with leukemogenic oncornaviruses. They are thought to be exfoliated viral-specified nonvirion cell surface antigens and have the property of adsorbing onto viable indicator cells, rendering the cells reactive to specific antisera in cytotoxic or immunofluorescence tests.
CLINICAL IMMUNOLOGY OF FELINE ONCORNA VIRUSES Basic immunologic studies of the FeLV are rapidly increasing our knowledge of the virus and its prevalence in the feline population. Employing immunologic methods, we have demonstrated that: (I) FeLV possesses a unique species specific antigen (gs-l) which distinguishes feline oncornaviruses from oncornaviruses of other species; (2) FeLV possesses an interspecies antigen (gs-3) which is shared by all mammalian leukemogenic oncornaviruses; (3) FeL V is widespread in the feline population; (4) FeLV is concentrated in the salivary glands of infected cats; (5) FeL V is associated with nonregenerative anemias of cats; (6) FeL V is horizontally (infectiously) spread; and (7) some cats are highly immune to FeLV due to neutralizing antibodies. The ultimate aim of our studies is to develop an effective vaccine against Fe LV and thus FeL V associated diseases.
Table 4.
Feline Oncornavirus Antigens
ANTIGENS
Virion antigens Envelope Internal
Cell surface antigens Viral-specified non~virion antigen Soluble antigens
SPECIFICITY
Type-specific Fe LV subgroups A, B, C Group-specific (gs) gs-l: species specific antigen gs-3 : interspecies (shared) antigen 3 other gs antigens present FOCMA (feline oncornavirus associated cell membrane antigen) Undefined in the feline leukemia virus system
140
WILLIAM
D.
HARDY, JR.
FEL V ANTIGENS Envelope Antigens Antigens of the viral envelope are coded for by the viral RNA and are assembled at the plasma membrane of the infected cell. Three subgroups of feline oncornavirus envelope antigens have been described; A, B, and CY Thus, the envelope antigens of subgroups A, B, and C are distinct (type-specific), giving rise to 3 Fe LV strains. Most of the FeLV isolates obtained from naturally occurring cases of lymphosarcoma consist of a mixture of subgroup A and B viruses. It may be possible for a cat to be immune to one subgroup of FeL V but susceptible to infection with other FeLV subgroups. FeSV isolates also fall into the 3 feline oncornavirus subgroups. The host range of any virus is governed by the viral envelope. Cells with specific cell receptor sites, complementary for an infecting virus, are susceptible to infection by that virus. Subgroups A, B, and C FeL V can infect cat fibroblasts in tissue culture. Recently, it has been shown that subgroup A FeLV was unable to infect cultured human cells while subgroups Band C FeL V were. It will be important to determine whether certain FeL V subgroups are responsible for specific feline diseases (see Table 2). In other words, subgroup A FeLV may induce lymphosarcoma while subgroup B FeLV may cause myeloproliferative disease and subgroup C FeLV may be responsible for nonregenerative anemias. Internal Antigens In contrast to the type-specific antigens of the viral envelope, the internal virion antigens are identical for the oncornaviruses of one species (group-specific). All subgroups of Fe LV and FeSV have identical gs antigens. 5 ,6 Gs antigens are valuable markers for detection of oncornavirus infection. The species of origin of any oncornavirus can be determined by the specificity of the gs-l (species-specific) antigen. The gs-l specificity is unique for feline oncornaviruses and this specificity differs from the oncornaviruses of other species. Oncornavirus gs antigens are produced in great excess in the cytoplasm of infected cells and a small amount of these antigens is assembled into the oncornavirus particle. Detection of FeLV infection utilizing antisera prepared against FeLV gs antigens is possible. We have developed a rapid, practical and sensitive fluorescent antibody test for detection of FeLV in cats. Figure 2 illustrates the detection of FeL V gs antigen in leukocytes and platelets from a peripheral blood smear of an infected cat. Utilizing this test, we have been able to show that the FeLV is transmitted infectiously between cats. The virus is passed mainly through the saliva and urine of infected cats. Table 5 shows that normal cats living with Fe LV positive cats have ;; 33 per cent chance of becoming infected. Twenty-five percent of FeLv
UNOLOGY OF ONCORNAVIRUSES
141
Figure 2. The fluorescent antibody tests in cats: top, leukocytes in a peripheral blood from a FeLV negative normal cat; bottom, cytoplasmic FeLV gs antigen fluorescence ranulocytes, lymphocytes, and platelets from a cat with naturally occurring Iympho-
lr
142
WILLIAM
Table 5.
D.
HARDY, JR .
FeLV Status of Normal Cats Related to Their Environment PER CENT OF ENVIRONMENT
Multiple cat households Previous FeLV positive cat No previous FeLV positive cat Single cat households Stray cats Cat colonies
FeLV
INFECTED NORMAL CATS
33
o
o
0.14
o
infected normal cats develop lymphosarcoma or a nonregenerative anemia within 6 months of FeL V detection. All mammalian leukemogenic and sarcomogenic oncornaviruses share a common gs-antigen (gs-3 or interspecies antigen).4 This common interspecies antigenic determinant is located on the same molecule as the species-specific gs-l determinant. The interspecies antigen is significant in that its detection in species where oncornaviruses have not yet been discovered, such as the dog and man, may lead to isolation of new oncornaVlruses.
Cell Surface Anitgens - FOCMA Many changes occur in cells infected with oncornaviruses, one of which is the induction of a new cell surface antigen (see Fig. 1). This antigen is specified by the infecting oncornavirus and appears in the cell membrane of the infected cell as a consequence of this infection. Feline, canine, and human cells infected with feline oncornaviruses produce a feline oncornavirus-associated cell membrane antigen (FOCMA).3 This antigen is not a structural component of the virion but is a new cell membrane antigen whose specificity is directed by the virus and not the infected cell. Cats infected with FeL V can produce specific antibody against the new cell surface antigen (FOCMA). Anti-FOCMA antibody will not neutralize FeL V but has specificity against the FOCMA of the infected cell surface. Under experimental conditions, cats that produce high FOCMA antibody do not develop progressing tumors. Thus, it appears that a high FOCMA antibody titer enables cats to resist, or even to reject, their viral induced tumors.
Soluble Antigens The soluble antigens, thought to be exfoliated viral-specified nonvirion cell surface antigens, have not been demonstrated nor studied in the feline system. This class of antigens seems to have little significance in the murine leukemia virus system.
143
IMMUNOLOGY OF ONCORNAVIRUSES
IMMUNOLOGIC RESPONSE OF CATS TO FeLV FeLV has an immunosuppressive effect as indicated by extended skin graft survival times in infected cats. The immunosuppressive action appears to affect the cellular rather than humoral component of the immune system. In general, cats can produce antibodies to FeL V antigens as well as to the viral-specified nonvirion cell surface antigen (FOCMA). Some cats under natural conditions produce high antiviral envelope (neutralizing) antibody titers and are highly resistant to FeL V infection. These cats probably have been exposed to low doses of FeL V and were able to mount an adequate humoral immune response. Unfortunately, most pet cats do not have protective neutralizing antibody titers and it is apparent that such cats are highly susceptible to FeL V infection. The use of communal litter pans and the unique social habits of cats are important factors in the spread of this virus. Cats can produce antibodies against FeL V gs antigens but the titers are quite low. Very sensitive techniques are required to demonstrate these antibodies. The significance of these antibodies is not well understood. Antibodies against gs-antigens do not neutralize infectious FeLV nor do they protect cats from the abnormal multiplication of infected cells as do anti-FOCMA antibodies. In fact, antibodies against gs antigens may even be detrimental. We have found gs antigen-antibody complexes in the glomeruli of some FeL V infected cats. Such antigenantibody complexes may be responsible for an immune-mediated glomerular injury in these cats. Cats do produce anti-FOCMA antibodies under natural conditions. High FOCMA antibody titers appear to be capable of protecting cats against developing lymphosarcoma. Table 6 summarizes six classes of
Table 6.
Classes of Cats Based on FeLV Status and Neutralizing and FOCMA Antibodies FeLV
CLASS OF CAT
Susceptible to Fe LV and lymphosarcoma Susceptible to Fe LV but resistant to lymphosarcoma Resistant to FeL V but susceptible to lymphosarcoma Resistant to FeLV and lymphosarcoma Very susceptible to develop lymphosarcoma Fe LV carrier but resistant to lymphosarcoma
FeLV
NEUTRALIZING
FOCMA
STATUS
ANTIBODY
ANTIBODY
+ + +
+
+ +
+
144
WILLIAM
D.
HARDY , JR.
cats based on their FeLV status, as well as their neutralizing and FOCMA antibody responses. We have found high neutralizing antibody with no FOCMA antibody in certain cats. Cats with high FOCMA antibody but no neutralizing antibodies have also been identified. Those FeL V negative cats who lack neutralizing and FOCMA antibodies are highly susceptible to FeL V infection and to de.velopment of lymphosarcoma. On the other hand, FeL V negative cats with high neutralizing and high FOCMA antibody titers are resistant to FeL V infection and to development of lymphosarcoma. FeL V infected cats do not have detectable neutralizing antibody. These cats will rapidly develop lymphosarcoma if they do not have a high FOCMA antibody titer. FeL V infected cats with high FOCMA antibody titers are resistant to development of lymphosarcoma and are thus inapparent healthy carriers shedding FeL V continuously in their saliva and urine.
IMMUNOLOGIC CONTROL OF FeL V RELATED DISEASES The ultimate control of FeLV related diseases would be the development of an FeL V vaccine. Such a vaccine seems feasible within the next few years. Since there are three subgroups of FeLV, all three would have to be included in any FeL V vaccine unless one or two of the subgroups were shown to be nonpathogenic. All of the cell culture, immunologic and epidemiologic techniques required to develop such a vaccineare now available. A modified live virus vaccine may be more effective than a killed vaccine in preventing FeLV related diseases. Live FeLV would replicate in inoculated cats and would thus induce FOCMA and the corresponding anti-FOCMA antibody. Anti-FOCMA antibody is extremely important in protecting against the development of FeLV related diseases. The use of a modified live FeLV vaccine in pet cats certainly poses several questions. Is it advisable to vaccinate cats with a live Fe LV which may be capable of infecting human cells? What consequences will the FeLV RNA have once it enters the cell? Will vaccinated cats shed infectious FeL V in their saliva and urine? Is there a possibility of a virulent FeL V mutant developing from the vaccine virus? Certainly most of these problems would be eliminated by the use of a killed virus vaccine. If a killed virus vaccine could induce a high FeLV neutralizing antibod} titer, then the need for anti-FOCMA antibodies would seem unneces· sary. At the present time what FeLV control measures are available to th( practicing veterinarian? First is the recognition that FeLV is spread in fectiously between cats. FeL V -carrying cats and cats suspected of carry
IMMUNOLOGY OF ONCORNAVIRUSES
145
ing the virus should be kept away from all other cats while hospitalized. The fluorescent antibody test for detection of FeL V in peripheral leukocytes is now available commercially. FeLV positive sick or normal carrier cats should be euthanatized since we do not yet know if this virus infects man. Until this question is answered, I feel the possible public health aspect of FeL V infected cats warrants their destruction , especially the sick cat whose illness is most certainly incurable. If an owner is reluctant to euthanatize an infected cat, then isolation is recommended. All remaining cats in the household should immediately be tested for FeLV. Any FeL V positive carrier cats should also be euthanatized or at least isolated. All negative cats should be retested in 3 months due to the long incubation period of the virus and the possibility of recent FeLV infection, which would not yet be apparent in the peripheral blood. By removing infected cats from contact with uninfected cats, we have been able to reduce the incidence of FeL V spread in multiple cat households and catteries. Only FeLV negative cats should be used for breeding purposes as FeL V can be transmitted in utero and through the milk. Ordinary household and hospital detergents are capable of inactivating FeL V in infected premises. At the present time the use of antiviral agents for therapy has not been explored. It is incumbent on the veterinarian to make a rapid and accurate diagnosis of FeLV related diseases and to recommend the proper course of action.
REFERENCES J. Brodey, R. S., McDonough, S. K., Frye, F. L. , and Hardy, W. D. , Jr.: Epidemiology of feline leukemia (lymphosarcoma). In Proceedings of the IV International Leukemia Symposium, Cherry Hill, 1969. Bib!. Haemat. 36:333, 1970. 2. Dorn, C. R., Taylor, D. O. N., and Hibbard, H. H. : Epizootiologic characteristics of canine and feline leukemia and lymphoma. A.j.V.R., 28:993, 1967. 3. Essex, M., Klein, G., Snyder, S. P. , and Harrold, j. B.: Feline sarcoma virus induced tumors: correlation between humoral antibody and tumor regression. Nature, 233: 195, 1971. 4. Geering, G., Aoki, T. , and Old, L. j. : Shared viral antigen of mammalian leukemia viruses. Nature, 226:265, 1970. 5. Hardy, W. D., Jr.: Immunodiffusion studies of feline leukemia and sarcoma. j.A.V.M.A., 158:1060, 197J. 6. Hardy, W. D. , Jr.: Feline lymphosarcoma: a model of viral carcinogenesis and significance related to human neoplasia. Animal Models for Biomedical Research IV . Washington, D. C., National Academy of Sciences, 197J. 7. Hardy, W. D., Jr. , Hirshaut, Y., and Hess, P. : Detection of the feline le ukemia virus and other mammalian oncornaviruses by immunofluorescence. In Proceedings of the 5th International Leukemia Symposium, Padova, Italy, Sept., 197 J. Bib!. Haemat. (in press). 8. Hardy, W. D., Jr., Old, L. j., Hess, P. W., Essex, M., and Cotter, S.: Horizontal transmission of feline leukemia virus; a field study. Nature, 244:266, 1973. 9. Jarrett, W. F. H ., Crawford, E. M., Martin , W. B., and Davie, F. : Leukemia in the cat. A virus-like particle associated with leukemia (lymphosarcoma). Nature, 202 :566, 1964.
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HARDY, JR.
10. Nowinski, R. C .. Old, L. J:. Sarkar. N. H .• and Moore, D. H .: Common properties of the oncogenic RNA viruses (oncornaviruses). Viro!.. 42 : 1152. 1970. II. Sarma. P. S .• and Log, T. : Viral interference in feline leukemia-sarcoma complex. Viro!. . 44:352.1971. 12. Snyder. S. P., and Theilen , G. H .: Transmissible feline fibrosarcoma. Nature. 221:1074. 1969. Laboratory of Veterinary Oncology Memorial-Sloan Kettering Cancer Center New York, New York
Immunology of Oncornaviruses
William D. Hardy, Jr., V.M.D.*
Virtually all organisms can be parasitized by viruses: vertebrate and invertebrate animals, plants, fungi, and bacteria. Some viruses are ubiquitous but are not known to cause a specific disease while other viruses cause diseases ranging from mild to fatal syndromes. Viruses which cause cancer are termed "oncogenic viruses." Both RNA and DNA viruses are etiologic agents of naturally occurring neoplasms in various animal species (Table 1). RNA viruses that cause naturally occurring neoplasms are found in chickens, cats, hamsters, mice, and primates. DNA viruses that cause naturally occurring neoplasms are found in frogs, chickens, rabbits, squirrels, dogs, cows, and even in man. The most significant oncogenic DNA viruses are the herpesviruses which cause leukemia in primates (Herpesvirus saimiri), renal adenocarcinomas in frogs, and neural lymphomatosis (Marek's disease) in chickens. The first oncogenic virus vaccine has recently been developed against the Marek's disease virus of chickens. This article will deal with the oncogenic RNA viruses and will specifically discuss the immunology of the feline oncogenic RNA viruses as important examples of these viruses in veterinary medicine. The feline leukemia and sarcoma viruses cause naturally occurring cancer in pet cats. 9 The feline sarcoma virus (FeSV) is an immunologically indistinguishable mutant of the feline leukemia virus (FeLV). FeSV causes malignant transformation of fibroblasts while FeLV causes malignant transformation of lymphoid cells (Table 2).12 FeL V is also associated with, and is thought to be the etiologic agent of, myeloproliferative disorders and nonregenerative anemias of cats.7 The cat has the highest incidence of lymphoreticular malignancies of any animal. Lymphosarcoma accounts for 90 per cent of all feline
*Associate,
Head; Laboratory of Veterinary Oncology, Memorial-Sloan Kettering Cancer Center, New York, New York; Consultant in Oncology, Henry Bergh Memorial Hospital of the American Society for Prevention of Cruelty to Animals and The Animal Medical Center, New York, New York.
Veterinary Clinics of NOlth America- Vol. 4, No. I, February 1974
133
134
WILLIAM
Table 1.
D.
HARDY, JR .
Oncogenic Viruses Causing Naturally Occurring Neoplasms DNA VIRUSES
RNA VIRUSES
Oncornaviruses Avian leukosis virus Avian myeloblastosis virus Avian erythroblastosis virus Avian sarcoma virus Avian reticuloendotheliosis virus Feline leukemia virus Feline sarcoma virus Hamster leukemia virus Murine leukemia virus (Gross) Murine mammary tumor virus Murine osteosarcoma virus Simian sarcoma virus (Wolly monkey)
Herpesviruses Herpesvirus saimiri (primate) Lucke tumor virus (frogs-renal adenocarcinoma) Marek's disease virus (neural lymphomatosis of chickens) Papovaviruses Bovine papilloma virus Canine oral papilloma virus Deer fibroma virus Human wart virus Rabbit oral papilloma virus Shope papilloma virus Poxviruses Hare fibroma virus Rabbit fibroma virus Rabbit myxoma virus Squirrel fibroma virus Yaba tumor virus
hematopoietic neoplasms. The annual incidence of lymphosarcoma is 41.6 cases per 100,000 cats in the population at risk, and represents onethird of all feline neoplasms.2 Thus, the FeL V is a very important virus in clinical veterinary medicine.
ONCORNA VIRUSES There are marked similarities among oncogenic RN A viruses which distinguish them from other RNA viruses. Previous designations for these viruses were "Ieukoviruses," "B-type and C-type particles," and Thylaxoviridae (sac-like viruses). However, none of these related to the most outstanding property of these viruses, namely, their ability to induce malignant transformation in the cells they infect. Therefore, the term "oncornavirus" (onco, "oncogenic" + rna, "ribonucleic acid") seems the most appropriate term to apply to this class of oncogenic RNA viruses.1o Table 3 lists the known oncornaviruses with their species of origin and notes if their oncogenicity has been proven. Oncornaviruses are widespread in the vertebrate phylum from reptiles to primates. Oncornaviruses are extremely important in veterinary medicine since the avian and feline oncornaviruses are the etiologic agents of the majority of cancer cases in these species. They have been found in association with bovine lymphosarcoma but their oncogenic potential still remains to be elucidated. Recently, oncornaviruses have been
.... ~ ~
c:
z o
5Cl ><:
o ." Table 2.
oz
Diseases Associated with Feline Oncornaviruses
C"l
o :
Feline Leukemia Virus
> ~Mutant
Feline Sarcoma Virus
<:
;; c:VJ t'1
VJ
1
Fibroblast Erythroid cell
Lymphoreticular cell
1
Lymphosarcoma or Reticulum cell sarcoma
~
Nonregenerative anemias
I
Myeloid cell ~
Erythroleukemia or Erythremic myelosis
1
1
Fibrosarcoma
Reticuloendotheliosis or Myelogenous leukemia
....
~
(J1
136
WILLIAM
Table 3.
D. HARDY, JR.
Occurrence of Oncornaviruses in Animals ONCOGENICITY OF
ANIMAL
ONCORNAVIRUS
ONCORNAVIRUS
FOUND
PROVEN
Reptiles Snake
+
Avian Chicken
+
+
Mammals Rodents Mouse Rat Guinea pig Hamster
+ + + +
+
Cow Pig Cat
+ + +
Lower primates Rhesus monkey Wolly monkey Gibbon ape
+ + +
Man
+
+ +
+
found in the Rhesus monkey, Wolly monkey, and Gibbon ape. The oncogenic capabilities of the Wolly monkey sarcoma virus have been demonstrated and it only appears a matter of time until human oncornaviruses are discovered.
BIOCHEMICAL PROPERTIES OF ONCORNAVIRUSES
I. All oncornaviruses have a 60-70S RNA. These fast sedimenting RNA's can be converted to 36-37S components by heating or by treatment with dimethylsulfoxide. Such a large viral RNA strand and the conversion of an RNA to a lower sedimentation coefficient are not characteristic of non-oncogenic RNA viruses. 2. In their native state the oncornaviruses do not have hemagglutinating activity. 3. In contrast to non-oncogenic RNA viruses, the production of oncornaviruses can be inhibited, in vitro, by actinomycin D. Since actinomycin D inhibits DNA synthesis, it was hard to understand why this drug should inhibit the production of RNA oncogenic viruses. Within the last 2 years this paradox has been explained by the monumental discovery that oncornaviruses initiate DNA synthesis specified by their viral RNA. The re-
IMMUNOLOGY OF ONCORNAVIRUSES
137
. action is mediated by an oncornavirus reverse transcriptase enzyme (RNA dependent DNA polymerase). This discovery has modified the central dogma of molecular biology which stated that DNA directed RN A synthesis. 4 . Oncornaviruses possess an enzyme, RNA dependent DNA polymerase, that produces DNA from viral RNA template. This enzyme enables oncornaviruses to insert a DNA code, specified by viral RNA templates, into the cellular chromosomes (cellular genome) . This new information may then be passed to subsequent cell generations. In the case of germinal cells, passage of viral-specified DNA to the next generation may result in vertical transmission of viral information.
BIOLOGICAL PROPERTIES OF ONCORNAVIRUSES
1. Oncornaviruses are usually oncogenic in their natural hosts. Three categories of neoplasms are induced by oncornaviruses: (1) leukemias, (2) sarcomas, and (3) mammary tumors (mouse and Rhesus monkey). These viruses may also cause other types of neoplasms as well as non-neoplastic diseases. 2. Oncornaviruses are widespread throughout the vertebrate phylum from reptiles to primates. 3 . Some oncornaviruses are transmitted chiefly vertically (murine oncornaviruses), while others are transmitted chiefly horizontally (feline oncornaviruses) . Vertical transmission occurs when oncornavirus RNA specifies DNA (virogene) which is then integrated into the chromosomes. Oncornaviruses can be activated after birth by activating the virogene (DNA) which produces viral RNA and viral particles. Until recently, it was thought that most oncornaviruses were transmitted exclusively vertically, but our findings of horizontal transmission of FeLV in cats have altered this concept. I, 8 4. Oncornaviruses are not cytopathic. This characteristic allows permanently infected virus-producing cell lines to be established. In animals, these viruses do not kill the cells in which they replicate. The neoplastically transformed infected cells continue to multiply, thus producing tumors.
CLASSES OF ONCORNA VIRUS ANTIGENS Three classes of antigens are associated with oncornaviruses and the cells they infect (Fig. 1 and Table 4). These antigens are (1) virion antigens, (2) cell surface antigens, and (3) soluble antigens.
Virion Antigens There are two types of virion antigens: (1) envelope (type-specific) lipoprotein antigens which are derived from the infected cell mem-
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INFECTED CELL VIRION ANTIGENS
Envelope antigens
o
~
.______@'--I 1.......' ..._ (group-specific)
Viral envelope } CELL SURFACE ANTIGENS
.
..............Viral sPf!C!fied ~ non-VIrion
·:.A~:\}:~luble antigens Figure 1.
Antigens associated with oncornaviruses.
brane, and (2) internal (group-specific) antigens located inside the VIrIon.
Envelope antigens are demonstrable by neutralization of infectivity in vitro or in vivo by complement fixation, immunodiffusion, viral interference tests and by immuno-electromicroscopy with visually labeled antibody (hybrid antibody). The demonstration of internal virion or group-specific (gs) antigens by immunodiffusion, complement fixation, and the paired radioiodine labeled antibody tests requires disruption of the viral envelope. Detection of gs antigens in infected cells requires destruction of the cell membrane by acetone fixation to allow penetration of specific antibody into the cytoplasm, the site of synthesis of these antigens. Cell Surface Antigens Cell surface antigens of oncornavirus infected cells are: (1) viral envelope components incorporated in the plasma membrane during the process of viral maturation, and (2) viral-specified cell surface components which are nonstructural elements of the virion. The former cell surface antigens are detectable by immuno-electronmicroscopy. Viral specified non virion cell surface antigens are analogous to the tumor-
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IMMUNOLOGY OF ONCORNAVIRUSES
specific transplantation antigens (TST A) specified by DNA oncogenic viruses. These antigens are demonstrable by immunofluorescence, immuno-electromicroscopy, and the cytotoxic test.
Soluble Antigens Soluble antigens are the third class of antigens and pervade the body fluids of animals infected with leukemogenic oncornaviruses. They are thought to be exfoliated viral-specified nonvirion cell surface antigens and have the property of adsorbing onto viable indicator cells, rendering the cells reactive to specific antisera in cytotoxic or immunofluorescence tests.
CLINICAL IMMUNOLOGY OF FELINE ONCORNA VIRUSES Basic immunologic studies of the FeLV are rapidly increasing our knowledge of the virus and its prevalence in the feline population. Employing immunologic methods, we have demonstrated that: (I) FeLV possesses a unique species specific antigen (gs-l) which distinguishes feline oncornaviruses from oncornaviruses of other species; (2) FeLV possesses an interspecies antigen (gs-3) which is shared by all mammalian leukemogenic oncornaviruses; (3) FeL V is widespread in the feline population; (4) FeLV is concentrated in the salivary glands of infected cats; (5) FeL V is associated with nonregenerative anemias of cats; (6) FeL V is horizontally (infectiously) spread; and (7) some cats are highly immune to FeLV due to neutralizing antibodies. The ultimate aim of our studies is to develop an effective vaccine against Fe LV and thus FeL V associated diseases.
Table 4.
Feline Oncornavirus Antigens
ANTIGENS
Virion antigens Envelope Internal
Cell surface antigens Viral-specified non~virion antigen Soluble antigens
SPECIFICITY
Type-specific Fe LV subgroups A, B, C Group-specific (gs) gs-l: species specific antigen gs-3 : interspecies (shared) antigen 3 other gs antigens present FOCMA (feline oncornavirus associated cell membrane antigen) Undefined in the feline leukemia virus system
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FEL V ANTIGENS Envelope Antigens Antigens of the viral envelope are coded for by the viral RNA and are assembled at the plasma membrane of the infected cell. Three subgroups of feline oncornavirus envelope antigens have been described; A, B, and CY Thus, the envelope antigens of subgroups A, B, and C are distinct (type-specific), giving rise to 3 Fe LV strains. Most of the FeLV isolates obtained from naturally occurring cases of lymphosarcoma consist of a mixture of subgroup A and B viruses. It may be possible for a cat to be immune to one subgroup of FeL V but susceptible to infection with other FeLV subgroups. FeSV isolates also fall into the 3 feline oncornavirus subgroups. The host range of any virus is governed by the viral envelope. Cells with specific cell receptor sites, complementary for an infecting virus, are susceptible to infection by that virus. Subgroups A, B, and C FeL V can infect cat fibroblasts in tissue culture. Recently, it has been shown that subgroup A FeLV was unable to infect cultured human cells while subgroups Band C FeL V were. It will be important to determine whether certain FeL V subgroups are responsible for specific feline diseases (see Table 2). In other words, subgroup A FeLV may induce lymphosarcoma while subgroup B FeLV may cause myeloproliferative disease and subgroup C FeLV may be responsible for nonregenerative anemias. Internal Antigens In contrast to the type-specific antigens of the viral envelope, the internal virion antigens are identical for the oncornaviruses of one species (group-specific). All subgroups of Fe LV and FeSV have identical gs antigens. 5 ,6 Gs antigens are valuable markers for detection of oncornavirus infection. The species of origin of any oncornavirus can be determined by the specificity of the gs-l (species-specific) antigen. The gs-l specificity is unique for feline oncornaviruses and this specificity differs from the oncornaviruses of other species. Oncornavirus gs antigens are produced in great excess in the cytoplasm of infected cells and a small amount of these antigens is assembled into the oncornavirus particle. Detection of FeLV infection utilizing antisera prepared against FeLV gs antigens is possible. We have developed a rapid, practical and sensitive fluorescent antibody test for detection of FeLV in cats. Figure 2 illustrates the detection of FeL V gs antigen in leukocytes and platelets from a peripheral blood smear of an infected cat. Utilizing this test, we have been able to show that the FeLV is transmitted infectiously between cats. The virus is passed mainly through the saliva and urine of infected cats. Table 5 shows that normal cats living with Fe LV positive cats have ;; 33 per cent chance of becoming infected. Twenty-five percent of FeLv
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141
Figure 2. The fluorescent antibody tests in cats: top, leukocytes in a peripheral blood from a FeLV negative normal cat; bottom, cytoplasmic FeLV gs antigen fluorescence ranulocytes, lymphocytes, and platelets from a cat with naturally occurring Iympho-
lr
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Table 5.
D.
HARDY, JR .
FeLV Status of Normal Cats Related to Their Environment PER CENT OF ENVIRONMENT
Multiple cat households Previous FeLV positive cat No previous FeLV positive cat Single cat households Stray cats Cat colonies
FeLV
INFECTED NORMAL CATS
33
o
o
0.14
o
infected normal cats develop lymphosarcoma or a nonregenerative anemia within 6 months of FeL V detection. All mammalian leukemogenic and sarcomogenic oncornaviruses share a common gs-antigen (gs-3 or interspecies antigen).4 This common interspecies antigenic determinant is located on the same molecule as the species-specific gs-l determinant. The interspecies antigen is significant in that its detection in species where oncornaviruses have not yet been discovered, such as the dog and man, may lead to isolation of new oncornaVlruses.
Cell Surface Anitgens - FOCMA Many changes occur in cells infected with oncornaviruses, one of which is the induction of a new cell surface antigen (see Fig. 1). This antigen is specified by the infecting oncornavirus and appears in the cell membrane of the infected cell as a consequence of this infection. Feline, canine, and human cells infected with feline oncornaviruses produce a feline oncornavirus-associated cell membrane antigen (FOCMA).3 This antigen is not a structural component of the virion but is a new cell membrane antigen whose specificity is directed by the virus and not the infected cell. Cats infected with FeL V can produce specific antibody against the new cell surface antigen (FOCMA). Anti-FOCMA antibody will not neutralize FeL V but has specificity against the FOCMA of the infected cell surface. Under experimental conditions, cats that produce high FOCMA antibody do not develop progressing tumors. Thus, it appears that a high FOCMA antibody titer enables cats to resist, or even to reject, their viral induced tumors.
Soluble Antigens The soluble antigens, thought to be exfoliated viral-specified nonvirion cell surface antigens, have not been demonstrated nor studied in the feline system. This class of antigens seems to have little significance in the murine leukemia virus system.
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IMMUNOLOGIC RESPONSE OF CATS TO FeLV FeLV has an immunosuppressive effect as indicated by extended skin graft survival times in infected cats. The immunosuppressive action appears to affect the cellular rather than humoral component of the immune system. In general, cats can produce antibodies to FeL V antigens as well as to the viral-specified nonvirion cell surface antigen (FOCMA). Some cats under natural conditions produce high antiviral envelope (neutralizing) antibody titers and are highly resistant to FeL V infection. These cats probably have been exposed to low doses of FeL V and were able to mount an adequate humoral immune response. Unfortunately, most pet cats do not have protective neutralizing antibody titers and it is apparent that such cats are highly susceptible to FeL V infection. The use of communal litter pans and the unique social habits of cats are important factors in the spread of this virus. Cats can produce antibodies against FeL V gs antigens but the titers are quite low. Very sensitive techniques are required to demonstrate these antibodies. The significance of these antibodies is not well understood. Antibodies against gs-antigens do not neutralize infectious FeLV nor do they protect cats from the abnormal multiplication of infected cells as do anti-FOCMA antibodies. In fact, antibodies against gs antigens may even be detrimental. We have found gs antigen-antibody complexes in the glomeruli of some FeL V infected cats. Such antigenantibody complexes may be responsible for an immune-mediated glomerular injury in these cats. Cats do produce anti-FOCMA antibodies under natural conditions. High FOCMA antibody titers appear to be capable of protecting cats against developing lymphosarcoma. Table 6 summarizes six classes of
Table 6.
Classes of Cats Based on FeLV Status and Neutralizing and FOCMA Antibodies FeLV
CLASS OF CAT
Susceptible to Fe LV and lymphosarcoma Susceptible to Fe LV but resistant to lymphosarcoma Resistant to FeL V but susceptible to lymphosarcoma Resistant to FeLV and lymphosarcoma Very susceptible to develop lymphosarcoma Fe LV carrier but resistant to lymphosarcoma
FeLV
NEUTRALIZING
FOCMA
STATUS
ANTIBODY
ANTIBODY
+ + +
+
+ +
+
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cats based on their FeLV status, as well as their neutralizing and FOCMA antibody responses. We have found high neutralizing antibody with no FOCMA antibody in certain cats. Cats with high FOCMA antibody but no neutralizing antibodies have also been identified. Those FeL V negative cats who lack neutralizing and FOCMA antibodies are highly susceptible to FeL V infection and to de.velopment of lymphosarcoma. On the other hand, FeL V negative cats with high neutralizing and high FOCMA antibody titers are resistant to FeL V infection and to development of lymphosarcoma. FeL V infected cats do not have detectable neutralizing antibody. These cats will rapidly develop lymphosarcoma if they do not have a high FOCMA antibody titer. FeL V infected cats with high FOCMA antibody titers are resistant to development of lymphosarcoma and are thus inapparent healthy carriers shedding FeL V continuously in their saliva and urine.
IMMUNOLOGIC CONTROL OF FeL V RELATED DISEASES The ultimate control of FeLV related diseases would be the development of an FeL V vaccine. Such a vaccine seems feasible within the next few years. Since there are three subgroups of FeLV, all three would have to be included in any FeL V vaccine unless one or two of the subgroups were shown to be nonpathogenic. All of the cell culture, immunologic and epidemiologic techniques required to develop such a vaccineare now available. A modified live virus vaccine may be more effective than a killed vaccine in preventing FeLV related diseases. Live FeLV would replicate in inoculated cats and would thus induce FOCMA and the corresponding anti-FOCMA antibody. Anti-FOCMA antibody is extremely important in protecting against the development of FeLV related diseases. The use of a modified live FeLV vaccine in pet cats certainly poses several questions. Is it advisable to vaccinate cats with a live Fe LV which may be capable of infecting human cells? What consequences will the FeLV RNA have once it enters the cell? Will vaccinated cats shed infectious FeL V in their saliva and urine? Is there a possibility of a virulent FeL V mutant developing from the vaccine virus? Certainly most of these problems would be eliminated by the use of a killed virus vaccine. If a killed virus vaccine could induce a high FeLV neutralizing antibod} titer, then the need for anti-FOCMA antibodies would seem unneces· sary. At the present time what FeLV control measures are available to th( practicing veterinarian? First is the recognition that FeLV is spread in fectiously between cats. FeL V -carrying cats and cats suspected of carry
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ing the virus should be kept away from all other cats while hospitalized. The fluorescent antibody test for detection of FeL V in peripheral leukocytes is now available commercially. FeLV positive sick or normal carrier cats should be euthanatized since we do not yet know if this virus infects man. Until this question is answered, I feel the possible public health aspect of FeL V infected cats warrants their destruction , especially the sick cat whose illness is most certainly incurable. If an owner is reluctant to euthanatize an infected cat, then isolation is recommended. All remaining cats in the household should immediately be tested for FeLV. Any FeL V positive carrier cats should also be euthanatized or at least isolated. All negative cats should be retested in 3 months due to the long incubation period of the virus and the possibility of recent FeLV infection, which would not yet be apparent in the peripheral blood. By removing infected cats from contact with uninfected cats, we have been able to reduce the incidence of FeL V spread in multiple cat households and catteries. Only FeLV negative cats should be used for breeding purposes as FeL V can be transmitted in utero and through the milk. Ordinary household and hospital detergents are capable of inactivating FeL V in infected premises. At the present time the use of antiviral agents for therapy has not been explored. It is incumbent on the veterinarian to make a rapid and accurate diagnosis of FeLV related diseases and to recommend the proper course of action.
REFERENCES J. Brodey, R. S., McDonough, S. K., Frye, F. L. , and Hardy, W. D. , Jr.: Epidemiology of feline leukemia (lymphosarcoma). In Proceedings of the IV International Leukemia Symposium, Cherry Hill, 1969. Bib!. Haemat. 36:333, 1970. 2. Dorn, C. R., Taylor, D. O. N., and Hibbard, H. H. : Epizootiologic characteristics of canine and feline leukemia and lymphoma. A.j.V.R., 28:993, 1967. 3. Essex, M., Klein, G., Snyder, S. P. , and Harrold, j. B.: Feline sarcoma virus induced tumors: correlation between humoral antibody and tumor regression. Nature, 233: 195, 1971. 4. Geering, G., Aoki, T. , and Old, L. j. : Shared viral antigen of mammalian leukemia viruses. Nature, 226:265, 1970. 5. Hardy, W. D., Jr.: Immunodiffusion studies of feline leukemia and sarcoma. j.A.V.M.A., 158:1060, 197J. 6. Hardy, W. D. , Jr.: Feline lymphosarcoma: a model of viral carcinogenesis and significance related to human neoplasia. Animal Models for Biomedical Research IV . Washington, D. C., National Academy of Sciences, 197J. 7. Hardy, W. D., Jr. , Hirshaut, Y., and Hess, P. : Detection of the feline le ukemia virus and other mammalian oncornaviruses by immunofluorescence. In Proceedings of the 5th International Leukemia Symposium, Padova, Italy, Sept., 197 J. Bib!. Haemat. (in press). 8. Hardy, W. D., Jr., Old, L. j., Hess, P. W., Essex, M., and Cotter, S.: Horizontal transmission of feline leukemia virus; a field study. Nature, 244:266, 1973. 9. Jarrett, W. F. H ., Crawford, E. M., Martin , W. B., and Davie, F. : Leukemia in the cat. A virus-like particle associated with leukemia (lymphosarcoma). Nature, 202 :566, 1964.
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10. Nowinski, R. C .. Old, L. J:. Sarkar. N. H .• and Moore, D. H .: Common properties of the oncogenic RNA viruses (oncornaviruses). Viro!.. 42 : 1152. 1970. II. Sarma. P. S .• and Log, T. : Viral interference in feline leukemia-sarcoma complex. Viro!. . 44:352.1971. 12. Snyder. S. P., and Theilen , G. H .: Transmissible feline fibrosarcoma. Nature. 221:1074. 1969. Laboratory of Veterinary Oncology Memorial-Sloan Kettering Cancer Center New York, New York