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Differential host range of viruses of feline leukemia-sarcoma complex
VIROLOGY 64, 438-446
Differential
(1975)
Host Range of Viruses
of Feline Leukemia-Sarcoma
Complex PADMAN Viral Carcinogenesis
S. SARMA, T. LOG, DAMINI JAIN, PAUL R. HILL, ROBERT J. HUEBNER Branch, National
Cancer Institute and Microbiological 20014 Accepted November
Associates,
AND
Bethesda, Maryland
20, 1974
Selected strains of feline leukemia and sarcoma viruses of subgroups A, B, and C show a differential pattern in their ability to cross species barrier and productively infect cells of heterologous host species. A virus of subgroup B showed the widest host range; it caused productive infection of cells of diverse host species including cells from cat, human, monkey, dog, bovine, pig, and hamster species. Two virus strains of subgroup A showed the narrowest host range; of the cells of several mammalian species examined, they only caused productive infection of cat and dog cells. Preliminary studies indicated that certain other strains of subgroup A viruses cause productive infection of whole human embryo cells. One strain of subgroup C virus examined showed a host range that was intermediate between that of A and B subgroup viruses. This strain caused productive infection of cat, dog, and certain human cells. In addition, the subgroup C virus caused productive infection of guinea pig cells found to be resistant to subgroup A as well as subgroup B virus strains examined. Preliminary studies suggested that certain, but not all, virus mixtures of A and B viruses can be purified into B type by passage of virus in heterologous human cells. The factor(s) that may govern the differential susceptibilities of heterologous host cells to the described strains of feline viruses are discussed. INTRODUCTION
The host range of avian leukosis and sarcoma viruses is determined by the viral envelope antigenic type of the virus. Avian cells such as chicken cells and quail cells exhibit genetically determined cellular susceptibility or resistance to one or more envelope antigenic types of virus (Vogt and Ishizaki, 1965; Payne and Biggs, 1964, 1966; Crittenden and Okazaki, 1965; Rubin, 1965; Hanafusa, 1965; Crittenden et al., 1967; Duff and Vogt, 1969; Payne et al., 1968, 1971). This property is believed to be governed by the presence or absence on the cell surface of receptors which permit the entry of a particular antigenic type of virus (Steck and Rubin, 1966a, 1966b; Piraino, 1967; Crittenden, 1968; Vogt, 1970). The ability of avian sarcoma viruses to cross species barrier and infect mammalian host cells was also shown to be dependent on the viral envelope antigenic type (Hana438 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
fusa and Hanafusa, 1966). Rous sarcoma virus pseudotype prepared with viral envelope of an avian leukosis virus RAV-50, a subgroup D virus (Duff and Vogt, 1969) was found to be the most pathogenic in this respect. We have found that feline cells of domestic cats from diverse sources do not exhibit partial or selective cellular resistance to any of the three envelope antigenic types of the feline leukemia and sarcoma viruses (Sarma and Log, 1973, Sarma et al., 1973a). However, cells of heterologous mammalian hosts exhibit susceptibility or partial or complete resistance to virus strains of different subgroups of feline leukosis virus. We describe herein our observations on this phenomenon. MATERIALS
AND METHODS
Tissue cultures. Cultures of feline embryo fibroblasts (FEF) used for the in vitro
FELINE LEUKEMIA-SARCOMA
propagation and assay of feline leukemia and sarcoma viruses were prepared and used as previously described (Sarma and Log, 1971, 1973; Sarma et al., 1971a, 1971b). Monolayer cultures of various other mammalian species were prepared or procured as follows: whole embryo cultures of human, canine, bovine, and porcine species and adult kidney cultures of Vero African green monkey were obtained from Microbiological Associates. We prepared monolayer cultures of inbred Strain 2 guinea pig embryo, hamster embryo, and Fischer rat embryo from whole embryos derived from animals at midterm in pregnancy. These animals were obtained from the Animal Production Section of the National Institutes of Health, Bethesda, MD. Other monolayer cultures were kindly provided as follows: NIH Swiss mouse embryo cultures by Dr. Janet W. Hartley, the RD line of human rhabdomyosarcoma cells (McAllister et al., 1971) by Dr. Robert M. McAllister, H37 suspension culture of human leukemic cells (lymphoblasts) by Dr. Paul Feorino and human skin and muscle culture by Dr. Raymond V. Gilden. The embryo-derived cultures were used between the 2nd and 10th in vitro passage levels. RD cultures were used between the 65th and 75th passages. All cultures were incubated at 37°C in a humidified CO, incubator flushed with 5% CO, in air and propagated in a medium prepared with Eagle’s minimum essential medium, supplemented with fetal calf serum and antibiotics, as described (Sarma and Log, 1971, 1973). Virus stocks. Feline leukemia virus (FeLV) of subgroups A, B, and C used were prepared as clarified culture fluids of FEF cultures productively infected with the following purified single antigenic type of virus (Sarma and Log, 1973). Two strains of subgroup A FeLV were used. These were the MAH (Sarma) strain and the 6%CT-7 (Gardner) strain. In addition, preliminary studies were also carried out with F-10 and FL-163 strains of subgroup A. One strain of FeLV of subgroup B was purified from a FeLV mixture of A and B present as associated virus in the Snyder-Theilen
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439
strain of feline sarcoma virus (Sarma et al., 1971b). One strain of FeLV of subgroup C was purified from FL-237 strain of FeLV, a mixture of A and C viruses (Sarma and Log, 1973). Purity of these FeLV of envelope antigenic types was repeatedly determined at periodic intervals by confirming that these FeLV strains induced homologous but not heterologous interference against challenge with focus-forming A, B, and C antigenic types of feline leukemia pseudotypes of murine sarcoma virus [MSV(FeLV)] (Sarma and Log, 1973). Virus stocks of uncloned field isolates of FeLV mixtures of A + B (several strains), A + C (strain FL-237), A + B + C (strain FL-74) were also similarly prepared. Virus stocks of Harvey MSV(FeLV) pseudotypes were prepared as clarified culture fluids of FEF confluently transformed with single antigenic types of virus. Test of host range. Replicate petri dish cultures of freshly plated heterologous host cells and homologous FEF cells were inoculated in parallel with 0.5-ml amounts of virus containing lo3 infectious units of each strain of FeLV. The inoculated and parallel control cultures were maintained by medium replacements at 3-day intervals and serial passages by trypsinization (Sarma et al., 1964) at weekly intervals. The cultures were periodically examined microscopically for morphological changes. Inoculated and control cultures were serially propagated. Replication of FeLV in inoculated homologous and heterologous cultures was determined at periodic intervals by examining the culture for the development of the group-specific (gs-1) antigen of the viruses of the feline leukemia-sarcoma group (COCAL test) (Sarma et al., 1971a). The induction of this antigen was determined by performing complement-fixation (CF) tests on cell antigens collected on day 21 and day 42 (in some cases, on day 63 as well) after virus inoculation. A virus was considered unable to infect cells of heterologous host species under the described conditions if the lo9 infectious units of virus caused a productive infection of homologous FEF within 21 days after virus inoculation but failed to cause a
440
SARMA
similar detectable infection (CF titer of cell antigens <1:2) in the heterologous culture on serial propagation of inoculated culture over a period of 42 days after virus inoculation. Test of the production of infectious virus in heterologous cultures. Culture fluids of virus-infected heterologous cultures were collected at the time of cell antigen collection between the 21st and 63rd day after virus inoculation, clarified by centrifugation at 2000 g for 20 min and tested for infectious virus either before or after storage at -70°C. Such fluids were inoculated in O.&ml amounts into replicate cultures of freshly plated FEF cells in disposable Falcon petri dishes. The envelope antigenic type of the virus thus reisolated was examined by viral interference test against challenge with focus-forming MSV(FeLV) of subgroups A, B, and C (Sarma and Log, 1973). of degree of susceptibility Determination of heterologous host cells in relation to homologous FEF cells. Certain heterologous host cells found susceptible to infection with FeLV were examined for their relative sensitivity to infection with the FeLV. The virus was assayed in parallel in such heterologous cultures and homologous FEF cultures by the CF antigen induction test (COCAL test) (Sarma et al., 1971a). The use of cells selectively resistant to subgroup A virus, for removal of subgroup A component from virus mixtures. Naturally occurring strains of FeLV of subgroups B and C are virus mixtures of A + B, A + C, or A + B + C (Sarma and Log, 1973). We attempted to determine if virus purification can be done by removal of the A component from virus mixtures by passage of virus mixtures through whole human embryo cells. Four virus isolates of A+B,oneofA+C,andoneofA+B+C were used (Sarma and Log, 1973). In addition, as control, we used two type A virus isolates we found to be infectious for human embryo cultures (strains F-10 and FL-163) and two A strains we found to be noninfectious for human embryo cultures (strain FL-237 purified from a virus mixture of A + C and strain F-6). Whole human cultures were productively infected
ET AL.
with 10’ infectious units of virus. The viral envelope antigenic type(s) of the progeny virus was determined by viral interference assay in FEF cultures as described (Sarma and Log, 1973). RESULTS
Results of three consecutive experiments summarized in Table 1 showed that ST-FeLV strain of FeLV of subgroup B had the widest host range. This virus thus established productive infection of a wide variety of mammalian cells including cells from dog, human, monkey, bovine, porcine, and hamster species. The virus failed to infect rodent cells such as cells of mouse, rat, and guinea pig (Table 1). One strain of FeLV of subgroup A (CT-7, Gardner) had the narrowest host range. This virus was infectious for only cat and dog cells. Another strain (Strain MAH, Sarma) was similarly found to infect cat cells but not four different human cultures. FL-237 strain of FeLV of subgroup C caused productive infection of cat and dog cells, and guinea pig cells which were resistant to subgroup A and B viruses. Under the conditions of our experiments, the FL-237 strain was infectious only for human tumor cells such as the human rhabdomyosarcoma cells, RD and human lymphoblasts H37. The virus failed to infect whole human embryo cultures and human skin and muscle cultures. All inoculated and control homologous and heterologous cultures failed to reveal cellular morphologic alterations over the entire duration of these experiments. Our recent preliminary studies indicate that F-10 and FL-163 strains of subgroup A FeLV cause productive infection of whole human embryo cells as well. Virus recovered from infected human cells was identified as type A virus by viral interference tests (Table 3). Dog cells we found to be susceptible to infection with CT-7 (Gardner) strain of subgroup A FeLV and FL-237 strain of subgroup C FeLV did not contain demonstrable gs-1 antigens of FeLV at 21 days after virus infection (CF titer < 1:2). However, clarified culture fluids collected at this time contained infectious FeLV capa-
TABLE DIFFERENTIAL
SUSCEPTIBILITIES
Virus
1
OF HETEROLOCOUS HOST CELLS TO INFECTION WITH FELINE LEUKEMIA SUBGROUPS A, B, AND Co
21
42
Cat Dog Guinea pig Hamster Mouse (Swiss) Rat (Fischer) Monkey (Vera) Bovine Human embryo Human skin and muscle RD (human) H 37 (human) Pig
>4’ <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2
>4 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2
FeLV, A MAH (Sarma)
Cat Human embryo Human skin and muscle H 37 (human) RD (human)
>4 <2 <2 <2 <2
<2 <2 <2 <2
FeLV, B (ST-FeLV)
Cat Dog Hamster Monkey (Vera) Bovine Human embryo Human skin and muscle RD (human) H 37 (human) Pig Guinea pig Mouse (Swiss) Rat (Fischer)
>4 >4 >4 >4 >4 >4 >4 >4 >4 >4 <2 <2 <2
Cat Dog Guinea pig RD (human) H 37 (human) Human embryo Human skin and muscle Hamster Mouse (Swiss) Rat (Fischer) Monkey (Vera) Bovine Pig Cat
>4 <2 >4 >4 >4 <2 <2 <2 <2 <2 <2 <2 <2 <2
FeLV, A 68-CT-7 (Gardner)
FeLV, C FL-237
Virus reisolation, FEP
Induction of gs-1 antigen on day
Host cell
VIRUSES OF
63
<2
<2
>4 >4 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 >4
>4 >4
>4 >4
>4 >4 <2 <2 <2
>4
>4 >4
>4
<2 <2 <2 <2 <2 <2 <2 <2 <2
<2
<2
>4 >4 >4 >4 >4 >4 >4 >4 >4 >4 <2 <2 <2 >4 >4 >4 >4 >4
<2 <2
<2 <2
a Cultures were infected with lo3 infectious units of virus. The induction of feline leukemia viral group-specific antigen was tested by complement-fixation test on cell antigens at various intervals. b Recovery of infectious virus from clarified culture fluids of infected cultures. c Reciprocal of highest antigen dilution giving a 3 to 4+ complement fixation against guinea pig antibodies directed against the gs-1 antigen of feline leukemia-sarcoma viruses. 441
442
SARMA ET AL.
TABLE 2 ble of inducing productive infection in OF THE RELATIVESUSCEPTIBILITIES OF homologous host (FEF) cultures. On serial A COMPARISON HETEROLOCOUS HOST CELLSAND HOMOLOGOUS CAT cell transfers of the infected dog cultures, CELLSTO INFECTIONWITHFELINELEUKEMIAVIRUSES FeLV gs-1 antigens, as well as infectious OF SUBGROUPS B AND C virus, were detected in demonstrable Virus” amounts on the 42nd and 63rd days after Host cell Highestvirus dilution virus infection (Table 1). inducing Other heterologous host cells we found to gs-1antig& be susceptible to the described FeLV FeLV A Cat embryo 10-S strains of subgroups B and C, developed (CT-7 Gardner) Dog embryo undiluted viral gs-1 antigen within 21 days after virus inoculation (CF titer of cell antigens 2 FeLV B Cat embryo 10-’ 1:4). On the 21st day, infectious FeLV was (ST-FeLV) Dog embryo lo-’ readily recovered from clarified culture Human embryo lo-’ fluids of all such ‘susceptible’ heterologous Monkey (Vera) lo-’ host cultures, including guinea pig and Bovine embryo lo-’ canine cultures (Table 1). The virus rePorcine embryo lo-’ covered from culture fluids of infected Cat embryo 10-a cultures replicated in FEF cultures and FeLV C (FL-237) Guinea pig embryo 10-S induced type specific viral interference against challenge with homologous 0 FeLV = feline leukemia virus. MSV( FeLV). Heterologous interference b Virus stocks containing 103-10’ infectious units of against other serotypes of MSV(FeLV) was feline leukemia virus were assayed in indicated culnot observed. tures. Cell antigens were collected on the 21st day
Relative susceptibilities of heterologous host cells. Dog cells were found to be
approximately lOOO-foldless sensitive than cat cells to infection with CT-7 (Gardner) strain of subgroup A virus (Table 2). On the other hand, dog cells as well as other susceptible heterologous host cells were as sensitive as homologous cat cells to infection with clone purified ST-FeLV of subgroup B. Similarly, guinea pig cells displayed the same degree of sensitivity as cat cells to infection with clone-purified FL-237 strain of FeLV of subgroup C. Removal or failure to remove subgroup A component from virus mixtures of A and B types. Two strains of subgroup A, strain
after virus inoculation and tested in the CF test for gs-1 antigen of FeLV (Sarma et al., 1971a).
A were identified as type A by viral interference tests (Sarma and Log, 1973). Human embryo cultures infected with A + B or A + B + C virus mixtures contained either B virus alone or a virus mixture of A and B. A reexamination of these inoculated human cultures for the antigenic type(s) of progeny virus gave the same results. The FL-237 strain of FeLV (mixture of A + C) failed to cause productive infection of whole human embryo cultures. As described above, purified single antigenic types of A and C derived from this strain similarly failed to infect the human embryo cells (Tables 1 and 3). As shown in Table 3, three FeLV mixtures of A + B viruses were purified into subgroup B virus by the described passage in whole human embryo cultures, whereas a similar passage of two other virus mixtures of A and B did not result in such a removal of the A virus components.
F-6 and strain FL-237 (purified from virus mixture of A + C), failed to infect the human embryo cells and were thus similar to strains CT-7 (Gardner) and MAH (Sarma). On the other hand, two single antigenie types of A virus (strains F-10 and FL-163), as well as antigenic mixtures of A + B and A + B + C viruses used in these studies induced productive infection of whole human embryo cells as determined DISCUSSION of CF antigen induction test (Sarma et al., Heterologous human and dog cells which 1971a). The progeny virus derived from cultures infected with single antigenic type we found to be fully susceptible to strain
FELINE LEUKEMIA-SARCOMACOMPLEX TABLE 3
443
virus. On the other hand, as described, we
REMOVALORFAILURETO REMOVETHE A VIRUSCOMPO-found that F-10 and FL-163 strains of NENT FROM VIRUS MIXTURES OF A AND. B BY subgroup A FeLV are able to cause producPASSAGE THROUGH WHOLE HUMAN EMBRYO tive infection of whole human embryo cells. CULTURES Further characterization of the complete
host range of these strains is presently in progress. Our preliminary studies suggest that certain virus mixtures of A and B types can be purified into subgroup B type by pasA A F-10 sage in whole human embryo cells suggestA A FL-163 ing that the subgroup A component present B F-4 A+B in these virus mixtures may not infect B FL-75 A+B whole human embryo cells. The support for B FL-165 A+B this contention comes from our preliminary ST-FeLV A+B A+B None observation that purified subgroup A FeLV FL-237 A+C FL-74 A+B A+B+C derived from one of these virus mixtures (strain F-4) of A and B viruses indeed does n Typing was done by viral interference tests in not infect human cells. On the other hand, feline embryo cultures (Sarma and Log, 1973). the passage in whole human embryo culST-FeLV of subgroup B and partially or tures of uncloned ST-FeLV (A + B types) completely resistant to two strains of sub- and uncloned FL-74 (A + B + C types) did group A [MAH (Sarma) and CT-7 not result in the elimination of the A (Gardner)], have been used by other inves- component present in these virus mixtures. tigators in the studies of feline sarcoma We believe that the A strains present in virus and their associated leukemia viruses these mixtures are infectious for whole (McAllister et al., 1973; Arnstein, P., per- human embryo cells. This observation will sonal communication, 1971). The original be confirmed with cloned subgroup A virus GA strain of feline sarcoma virus (GA- we are now in the process of preparing from FSV) and the accompanying leukemia these stocks. Thus, our observations sugvirus (GA-FeLV) used in these studies are gest that the recovery of both A and B mixtures of A and B viruses (Sarma et al., types by Jarrett et al. (1973) by passage of 1971b, 1971c). However, we found that their FeLV No. 5 through human cells can cells chronically infected with GA-FeLV, be attributed to the parallel infectivity for such as human rhabdomyosarcoma cells human cells of both A as well as B types RD (obtained from Dr. R. M. McAllister) present in their virus mixture rather than and beagle dog embryo cells (obtained due to entry of A type viral genome into from Dr. P. Arnstein) release only the human cells with B-type envelope (phenosubgroup B FeLV. This initial observation typic mixing) as originally believed (Jarsuggesting a partial or complete resistance rett et al., 1973). of human and dog cells to subgroup A virus We found that FL-237 strain of FeLV of led to the present studies. We found that subgroup C has a host range that is neither dog cells are partially resistant to CT-7 as wide as that of strain ST-FeLV of (Gardner) strain of subgroup A virus and subgroup B virus nor as narrow as that of that whole human cells and cells of many strain FL-237 of subgroup A virus. In different mammalian species fail to sup- repeated studies we found this strain to be port the replication of this strain. Our infectious for only certain, but not all, recent quantitative studies with relatively human cells. The FL-237 strain also large doses of CT-7 (Gardner) strain of showed a peculiar tropism for guinea pig subgroup A FeLV ( lo5 infectious units) has cells not displayed by the viruses of subshown that the insusceptibility of human groups A and B we examined. cells to this strain of subgroup A FeLV We found that in every case where puricannot be overcome with large doses of fied FeLV of subgroups A, B, or C caused Virus strain
Envelope antigenie types present
Envelope antigenic type(s) isolated after passage through human cells”
444
SARMA ET AL.
demonstrable virus infection in a heterologous host species, there was no host range modification of virus, demonstrable by a loss of viral infectivity for the homologous host cells, FEF. In addition, the viral envelope specificity demonstrable by viral interference tests was not modified by passage through such heterologous host cells. Jarrett et al. (1973) found that, of the cells of these three species they studied, FeLV of subgroup A was infectious only for cat cells, whereas FeLV of subgroups B and C infected cat, dog, as well as human cells. As described herein, we found that strain ST-FeLV of subgroup B has the widest host range, being able to cause productive infection of a variety of heterologous host cells including human and dog cells. We found that strain CT-7 (Gardner) of subgroup A strain has the narrowest host range. In addition to cat cells, this strain infected dog cells as well. More importantly, we found two strains (F-10 and FL-163) of subgroup A virus to be infectious for whole human embryo cells. In our hands, FL-237 strain of subgroup C virus, found to be infectious for human cells by Jarrett et al. (1973), failed to cause productive infection of certain normal human cells such as whole human embryo cells and human skin and muscle cells; thus, this virus cannot be considered as infectious for human cells as ST-FeLV strain of subgroup B we have described herein. In these studies we found interesting virus strain specific differences within A subgroup in their ability to infect human cells. One strain of subgroup C we studied revealed intraspecies specific differences within a host species (human). The factor(s) responsible for the differential sensitivities of different strains of FeLV are presently unknown. If viral envelope antigens are responsible for the observed differences, it is not readily apparent why subgroup A viruses show a differential pattern in their ability to cause productive infection of heterologous human cells. Minor envelope antigenic differences within members of a subgroup may exist which may account for observed differences in host range. This remains to
be determined. Further studies with additional strains of subgroup A, B, and C viruses are needed before seriously considering viral envelope antigens as being responsible for the observed differences in the host range of these viruses. While additional strains of subgroup A and B are available for this study, at present there exists only one known strain of subgroup C virus; the FL-237 strain and FL-74 strain of subgroup C were derived from the same source (Theilen, G. H., personal communication). It also remains to be determined whether the observed insusceptibility of certain heterologous host cells to FeLV is due to an intracellular block in virus replication as has been shown for N and B tropic mouse type C viruses in mouse cells of B and N types, respectively (Huang et al., 1973; Krontiris et al., 1973). Although these studies have only considered productive infection as a sign of infection, nonproductive infections of heterologous host cells as occurs when avian sarcoma viruses infect mammalian cells (Huebner et al., 1964) have to be considered. Whereas avian sarcoma viral gs antigens can be found in such mammalian cells (Huebner et al., 1964), we failed to find the gs antigens of FeLV detectable by CF test in heterologous cells we found to be insusceptible to productive infection with FeLV. Hardy et al. (1973) and Jarrett et al. (1973) recently showed that FeLV has a remarkable capacity to undergo horizontal transmission to uninfected cats under natural (Hardy et al., 1973) and experimental (Jarrett et al., 1973) conditions accounting for a significant proportion of the cancers of this domestic pet. Our findings on the host range of FeLV types, especially the wide host range of ST-FeLV strain of subgroup B, may have important implications in the natural spread of these viruses to other species. Our seroepidemiological studies of cats and humans have shown that FeLV of all three subgroups are widespread in domestic cats as evidence by the presence of significant levels of virus-neutralizing antibodies in cats with or without neoplasia (Sarma et al., 197313). However, humans failed to reveal the occurrence of overt
FELINE
LEUKEMIA-SARCOMA
natural infections with FeLV demonstrable by the development of virus-neutralizing serum antibodies in subjects in close contact with cat and/or FeLV, such as veterinarians and laboratory workers engaged in FeLV research. The feline leukemia viruses resemble the avian leukosis viruses in the phenomenon of type-specific viral interference (Steck and Rubin, 1966a, 1966b, and Vogt, 1970) and in their ability or inability to cross species barrier and infect cells of heterologous host species (Hanafusa, 1966). The presently discernible difference between the avian leukosis viruses and the feline leukemia-sarcoma viruses appears to be in the relative susceptibilities of homologous host cells to infection with the different antigenic types of virus; whereas, cat cells are uniformly susceptible to the three serotypes of FeLV we have described, the avian cells show a differential pattern of susceptibility or resistance to viruses of different avian leukosis subgroups (Vogt and Ishizaki, 1965; Crittenden, 1968; Payne et al., 1968; Duff and Vogt, 1969; Vogt, 1970). Further studies with cat cells from “inbred” cats may be necessary to determine whether cells from such cats may show differential susceptibilities to the described serotypes of FeLV. ACKNOWLEDGMENT This work NIH-NOl-CP-43254 of the National 20014.
was supported by Contract within the Virus Cancer Program Cancer Institute, Bethesda, MD REFERENCES
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USA 70, 2549-2553. MCALLISTER, R. M., NELSON-REES, W. A., JOHNSON, E. Y., RONGEY, R. W., and GARDNER, M. B. (1971). Disseminated rhabdomyosarcomas formed in kittens by cultured human rhabdomyosarcoma cells. J. Nat. Cancer Inst. 47, 603-607. MCALLISTER, R. M., NICOLSON, M., GARDNER, M. B., RASHEED, S., RONGEY, R. W., HARDY, W. D., and GILDEN, R. V. (1973). RD-114 virus compared with feline and murine type-C viruses released from RD cells. Nature New Biol. 242, 75-78. PAYNE, L. N., and BIGGS, P. M. (1964). Differences between highly inbred lines of chickens in the response to Rous sarcoma virus of the chorioallantoic membrane and of embryonic cells in tissue culture. Virology 24, 610-616. PAYNE, L. N., and BEGS, P. M. (1966). Genetic basis of cellular susceptibility to Schmidt-Ruppin and Harris strains of Rous sarcoma virus. Virology 29, 190-198. PAYNE, L. N., CRITTENDEN, L. B., and OKAZAKI, W. (1968). Influence of host genotype on responses to four strains of avian leukosis virus. J. Nat. Cancer Inst. 40, 907-916. PAYNE, L. N., PANI, P. K.. and WEISS, R. A. (1971). A dominant epistatic gene which inhibits cellular susceptibility to RSV(RAV-0). J. Gen. Virol. 13, 455-462.
SARMA PIRAINO, F. (1967). The mechanism of genetic resistance of chick embryo cells to infection by Rous sarcoma virus-Bryan strain (BS-RSV). Virology 32, 700-707. RUBIN, H. (1965). Genetic control of cellular susceptibility to pseudotypes of Rous sarcoma virus. Virology 26, 270-276. SARMA, P. S., TURNER, H. C., and HUEBNER, R. J. (1964). An avian leukosis group-specific complement fixation reaction. Application for the detection and assay of non-cytopathogenic leukosis viruses. Virology 23, 313-321. SARMA, P. S., and LOG, T. (1971). Viral interference in feline leukemia-sarcoma complex. Virology 44, 352-358. SARMA, P. S., GILDEN, R. V., and HUEBNER, R. J. (1971a). Complement-fixation test for feline leukemia and sarcoma viruses (the COCAL test). Virology 44, 137-145. SARMA, P. S., LOG, T., and THEILEN, G. H. (1971b). ST feline sarcoma virus. Biological characteristics and in vitro propagation. Proc. Sot. Exp. Biol. Med. 137, 1444-1448. SARMA, P. S., BASKAR, J. F., GILDEN, R. V., GARDNER, M. B., and HUEBNER, R. J. (1971c). In uitro isolation and characterization of the GA strain of feline sarcoma virus. Proc. Sot. Exp. Biol. Med. 137, 1333-1336. SARMA, P. S. and LOG, T. (1973). Subgroup classification of feline leukemia and sarcoma viruses by viral
ET AL. interference and neutralization tests. Virology 54, 160-169. SARMA, P. S., JAIN, D., and HILL, P. (1973a). In vitro host range of feline leukemia virus. Proc. Sixth Znt. Symp. Comp. Leukemia Res. 437-440, In Press. Univ. of Tokyo Press, Tokyo, Japan. SARMA, P. S., SHARAR, A., WALTERS, V., and GARDNER, M. (1973b). A survey of cats and humans for prevalence of feline leukemia-sarcoma virus neutralizing serum antibodies. Proc. Sot. Exp. Biol. Med. 145, 560-564. STECK, F. T., and RUBIN, H. (1966a). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. I. Establishment of interference. Virology 29, 628-641. STECK, F. T., and RUBIN, H. (1966b). The mechanism of interference between an avian leukosis virus and Rous sarcoma virus. II. Early steps of infection by RSV of cells under conditions of interference. Virology 29, 642-653. SNYDER, S. P., and THEILEN, G. H. (1969). Transmissible feline fibrosarcoma. Nature 221, 1074-1075. VOGT, P. K., and ISHIZAKI, R. (1965). Reciprocal patterns of genetic resistance to avian tumor viruses in two lines of chickens. Virology 26, 664-672. VOGT, P. K. (1970). Envelope classification of avian RNA tumor viruses. Fourth Int. Symp. Comp. Leuk. Res.. Biblia Hematol. 36, pp. 153-167. Karger, Basel.
Differential
(1975)
Host Range of Viruses
of Feline Leukemia-Sarcoma
Complex PADMAN Viral Carcinogenesis
S. SARMA, T. LOG, DAMINI JAIN, PAUL R. HILL, ROBERT J. HUEBNER Branch, National
Cancer Institute and Microbiological 20014 Accepted November
Associates,
AND
Bethesda, Maryland
20, 1974
Selected strains of feline leukemia and sarcoma viruses of subgroups A, B, and C show a differential pattern in their ability to cross species barrier and productively infect cells of heterologous host species. A virus of subgroup B showed the widest host range; it caused productive infection of cells of diverse host species including cells from cat, human, monkey, dog, bovine, pig, and hamster species. Two virus strains of subgroup A showed the narrowest host range; of the cells of several mammalian species examined, they only caused productive infection of cat and dog cells. Preliminary studies indicated that certain other strains of subgroup A viruses cause productive infection of whole human embryo cells. One strain of subgroup C virus examined showed a host range that was intermediate between that of A and B subgroup viruses. This strain caused productive infection of cat, dog, and certain human cells. In addition, the subgroup C virus caused productive infection of guinea pig cells found to be resistant to subgroup A as well as subgroup B virus strains examined. Preliminary studies suggested that certain, but not all, virus mixtures of A and B viruses can be purified into B type by passage of virus in heterologous human cells. The factor(s) that may govern the differential susceptibilities of heterologous host cells to the described strains of feline viruses are discussed. INTRODUCTION
The host range of avian leukosis and sarcoma viruses is determined by the viral envelope antigenic type of the virus. Avian cells such as chicken cells and quail cells exhibit genetically determined cellular susceptibility or resistance to one or more envelope antigenic types of virus (Vogt and Ishizaki, 1965; Payne and Biggs, 1964, 1966; Crittenden and Okazaki, 1965; Rubin, 1965; Hanafusa, 1965; Crittenden et al., 1967; Duff and Vogt, 1969; Payne et al., 1968, 1971). This property is believed to be governed by the presence or absence on the cell surface of receptors which permit the entry of a particular antigenic type of virus (Steck and Rubin, 1966a, 1966b; Piraino, 1967; Crittenden, 1968; Vogt, 1970). The ability of avian sarcoma viruses to cross species barrier and infect mammalian host cells was also shown to be dependent on the viral envelope antigenic type (Hana438 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
fusa and Hanafusa, 1966). Rous sarcoma virus pseudotype prepared with viral envelope of an avian leukosis virus RAV-50, a subgroup D virus (Duff and Vogt, 1969) was found to be the most pathogenic in this respect. We have found that feline cells of domestic cats from diverse sources do not exhibit partial or selective cellular resistance to any of the three envelope antigenic types of the feline leukemia and sarcoma viruses (Sarma and Log, 1973, Sarma et al., 1973a). However, cells of heterologous mammalian hosts exhibit susceptibility or partial or complete resistance to virus strains of different subgroups of feline leukosis virus. We describe herein our observations on this phenomenon. MATERIALS
AND METHODS
Tissue cultures. Cultures of feline embryo fibroblasts (FEF) used for the in vitro
FELINE LEUKEMIA-SARCOMA
propagation and assay of feline leukemia and sarcoma viruses were prepared and used as previously described (Sarma and Log, 1971, 1973; Sarma et al., 1971a, 1971b). Monolayer cultures of various other mammalian species were prepared or procured as follows: whole embryo cultures of human, canine, bovine, and porcine species and adult kidney cultures of Vero African green monkey were obtained from Microbiological Associates. We prepared monolayer cultures of inbred Strain 2 guinea pig embryo, hamster embryo, and Fischer rat embryo from whole embryos derived from animals at midterm in pregnancy. These animals were obtained from the Animal Production Section of the National Institutes of Health, Bethesda, MD. Other monolayer cultures were kindly provided as follows: NIH Swiss mouse embryo cultures by Dr. Janet W. Hartley, the RD line of human rhabdomyosarcoma cells (McAllister et al., 1971) by Dr. Robert M. McAllister, H37 suspension culture of human leukemic cells (lymphoblasts) by Dr. Paul Feorino and human skin and muscle culture by Dr. Raymond V. Gilden. The embryo-derived cultures were used between the 2nd and 10th in vitro passage levels. RD cultures were used between the 65th and 75th passages. All cultures were incubated at 37°C in a humidified CO, incubator flushed with 5% CO, in air and propagated in a medium prepared with Eagle’s minimum essential medium, supplemented with fetal calf serum and antibiotics, as described (Sarma and Log, 1971, 1973). Virus stocks. Feline leukemia virus (FeLV) of subgroups A, B, and C used were prepared as clarified culture fluids of FEF cultures productively infected with the following purified single antigenic type of virus (Sarma and Log, 1973). Two strains of subgroup A FeLV were used. These were the MAH (Sarma) strain and the 6%CT-7 (Gardner) strain. In addition, preliminary studies were also carried out with F-10 and FL-163 strains of subgroup A. One strain of FeLV of subgroup B was purified from a FeLV mixture of A and B present as associated virus in the Snyder-Theilen
COMPLEX
439
strain of feline sarcoma virus (Sarma et al., 1971b). One strain of FeLV of subgroup C was purified from FL-237 strain of FeLV, a mixture of A and C viruses (Sarma and Log, 1973). Purity of these FeLV of envelope antigenic types was repeatedly determined at periodic intervals by confirming that these FeLV strains induced homologous but not heterologous interference against challenge with focus-forming A, B, and C antigenic types of feline leukemia pseudotypes of murine sarcoma virus [MSV(FeLV)] (Sarma and Log, 1973). Virus stocks of uncloned field isolates of FeLV mixtures of A + B (several strains), A + C (strain FL-237), A + B + C (strain FL-74) were also similarly prepared. Virus stocks of Harvey MSV(FeLV) pseudotypes were prepared as clarified culture fluids of FEF confluently transformed with single antigenic types of virus. Test of host range. Replicate petri dish cultures of freshly plated heterologous host cells and homologous FEF cells were inoculated in parallel with 0.5-ml amounts of virus containing lo3 infectious units of each strain of FeLV. The inoculated and parallel control cultures were maintained by medium replacements at 3-day intervals and serial passages by trypsinization (Sarma et al., 1964) at weekly intervals. The cultures were periodically examined microscopically for morphological changes. Inoculated and control cultures were serially propagated. Replication of FeLV in inoculated homologous and heterologous cultures was determined at periodic intervals by examining the culture for the development of the group-specific (gs-1) antigen of the viruses of the feline leukemia-sarcoma group (COCAL test) (Sarma et al., 1971a). The induction of this antigen was determined by performing complement-fixation (CF) tests on cell antigens collected on day 21 and day 42 (in some cases, on day 63 as well) after virus inoculation. A virus was considered unable to infect cells of heterologous host species under the described conditions if the lo9 infectious units of virus caused a productive infection of homologous FEF within 21 days after virus inoculation but failed to cause a
440
SARMA
similar detectable infection (CF titer of cell antigens <1:2) in the heterologous culture on serial propagation of inoculated culture over a period of 42 days after virus inoculation. Test of the production of infectious virus in heterologous cultures. Culture fluids of virus-infected heterologous cultures were collected at the time of cell antigen collection between the 21st and 63rd day after virus inoculation, clarified by centrifugation at 2000 g for 20 min and tested for infectious virus either before or after storage at -70°C. Such fluids were inoculated in O.&ml amounts into replicate cultures of freshly plated FEF cells in disposable Falcon petri dishes. The envelope antigenic type of the virus thus reisolated was examined by viral interference test against challenge with focus-forming MSV(FeLV) of subgroups A, B, and C (Sarma and Log, 1973). of degree of susceptibility Determination of heterologous host cells in relation to homologous FEF cells. Certain heterologous host cells found susceptible to infection with FeLV were examined for their relative sensitivity to infection with the FeLV. The virus was assayed in parallel in such heterologous cultures and homologous FEF cultures by the CF antigen induction test (COCAL test) (Sarma et al., 1971a). The use of cells selectively resistant to subgroup A virus, for removal of subgroup A component from virus mixtures. Naturally occurring strains of FeLV of subgroups B and C are virus mixtures of A + B, A + C, or A + B + C (Sarma and Log, 1973). We attempted to determine if virus purification can be done by removal of the A component from virus mixtures by passage of virus mixtures through whole human embryo cells. Four virus isolates of A+B,oneofA+C,andoneofA+B+C were used (Sarma and Log, 1973). In addition, as control, we used two type A virus isolates we found to be infectious for human embryo cultures (strains F-10 and FL-163) and two A strains we found to be noninfectious for human embryo cultures (strain FL-237 purified from a virus mixture of A + C and strain F-6). Whole human cultures were productively infected
ET AL.
with 10’ infectious units of virus. The viral envelope antigenic type(s) of the progeny virus was determined by viral interference assay in FEF cultures as described (Sarma and Log, 1973). RESULTS
Results of three consecutive experiments summarized in Table 1 showed that ST-FeLV strain of FeLV of subgroup B had the widest host range. This virus thus established productive infection of a wide variety of mammalian cells including cells from dog, human, monkey, bovine, porcine, and hamster species. The virus failed to infect rodent cells such as cells of mouse, rat, and guinea pig (Table 1). One strain of FeLV of subgroup A (CT-7, Gardner) had the narrowest host range. This virus was infectious for only cat and dog cells. Another strain (Strain MAH, Sarma) was similarly found to infect cat cells but not four different human cultures. FL-237 strain of FeLV of subgroup C caused productive infection of cat and dog cells, and guinea pig cells which were resistant to subgroup A and B viruses. Under the conditions of our experiments, the FL-237 strain was infectious only for human tumor cells such as the human rhabdomyosarcoma cells, RD and human lymphoblasts H37. The virus failed to infect whole human embryo cultures and human skin and muscle cultures. All inoculated and control homologous and heterologous cultures failed to reveal cellular morphologic alterations over the entire duration of these experiments. Our recent preliminary studies indicate that F-10 and FL-163 strains of subgroup A FeLV cause productive infection of whole human embryo cells as well. Virus recovered from infected human cells was identified as type A virus by viral interference tests (Table 3). Dog cells we found to be susceptible to infection with CT-7 (Gardner) strain of subgroup A FeLV and FL-237 strain of subgroup C FeLV did not contain demonstrable gs-1 antigens of FeLV at 21 days after virus infection (CF titer < 1:2). However, clarified culture fluids collected at this time contained infectious FeLV capa-
TABLE DIFFERENTIAL
SUSCEPTIBILITIES
Virus
1
OF HETEROLOCOUS HOST CELLS TO INFECTION WITH FELINE LEUKEMIA SUBGROUPS A, B, AND Co
21
42
Cat Dog Guinea pig Hamster Mouse (Swiss) Rat (Fischer) Monkey (Vera) Bovine Human embryo Human skin and muscle RD (human) H 37 (human) Pig
>4’ <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2
>4 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2
FeLV, A MAH (Sarma)
Cat Human embryo Human skin and muscle H 37 (human) RD (human)
>4 <2 <2 <2 <2
<2 <2 <2 <2
FeLV, B (ST-FeLV)
Cat Dog Hamster Monkey (Vera) Bovine Human embryo Human skin and muscle RD (human) H 37 (human) Pig Guinea pig Mouse (Swiss) Rat (Fischer)
>4 >4 >4 >4 >4 >4 >4 >4 >4 >4 <2 <2 <2
Cat Dog Guinea pig RD (human) H 37 (human) Human embryo Human skin and muscle Hamster Mouse (Swiss) Rat (Fischer) Monkey (Vera) Bovine Pig Cat
>4 <2 >4 >4 >4 <2 <2 <2 <2 <2 <2 <2 <2 <2
FeLV, A 68-CT-7 (Gardner)
FeLV, C FL-237
Virus reisolation, FEP
Induction of gs-1 antigen on day
Host cell
VIRUSES OF
63
<2
<2
>4 >4 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 >4
>4 >4
>4 >4
>4 >4 <2 <2 <2
>4
>4 >4
>4
<2 <2 <2 <2 <2 <2 <2 <2 <2
<2
<2
>4 >4 >4 >4 >4 >4 >4 >4 >4 >4 <2 <2 <2 >4 >4 >4 >4 >4
<2 <2
<2 <2
a Cultures were infected with lo3 infectious units of virus. The induction of feline leukemia viral group-specific antigen was tested by complement-fixation test on cell antigens at various intervals. b Recovery of infectious virus from clarified culture fluids of infected cultures. c Reciprocal of highest antigen dilution giving a 3 to 4+ complement fixation against guinea pig antibodies directed against the gs-1 antigen of feline leukemia-sarcoma viruses. 441
442
SARMA ET AL.
TABLE 2 ble of inducing productive infection in OF THE RELATIVESUSCEPTIBILITIES OF homologous host (FEF) cultures. On serial A COMPARISON HETEROLOCOUS HOST CELLSAND HOMOLOGOUS CAT cell transfers of the infected dog cultures, CELLSTO INFECTIONWITHFELINELEUKEMIAVIRUSES FeLV gs-1 antigens, as well as infectious OF SUBGROUPS B AND C virus, were detected in demonstrable Virus” amounts on the 42nd and 63rd days after Host cell Highestvirus dilution virus infection (Table 1). inducing Other heterologous host cells we found to gs-1antig& be susceptible to the described FeLV FeLV A Cat embryo 10-S strains of subgroups B and C, developed (CT-7 Gardner) Dog embryo undiluted viral gs-1 antigen within 21 days after virus inoculation (CF titer of cell antigens 2 FeLV B Cat embryo 10-’ 1:4). On the 21st day, infectious FeLV was (ST-FeLV) Dog embryo lo-’ readily recovered from clarified culture Human embryo lo-’ fluids of all such ‘susceptible’ heterologous Monkey (Vera) lo-’ host cultures, including guinea pig and Bovine embryo lo-’ canine cultures (Table 1). The virus rePorcine embryo lo-’ covered from culture fluids of infected Cat embryo 10-a cultures replicated in FEF cultures and FeLV C (FL-237) Guinea pig embryo 10-S induced type specific viral interference against challenge with homologous 0 FeLV = feline leukemia virus. MSV( FeLV). Heterologous interference b Virus stocks containing 103-10’ infectious units of against other serotypes of MSV(FeLV) was feline leukemia virus were assayed in indicated culnot observed. tures. Cell antigens were collected on the 21st day
Relative susceptibilities of heterologous host cells. Dog cells were found to be
approximately lOOO-foldless sensitive than cat cells to infection with CT-7 (Gardner) strain of subgroup A virus (Table 2). On the other hand, dog cells as well as other susceptible heterologous host cells were as sensitive as homologous cat cells to infection with clone purified ST-FeLV of subgroup B. Similarly, guinea pig cells displayed the same degree of sensitivity as cat cells to infection with clone-purified FL-237 strain of FeLV of subgroup C. Removal or failure to remove subgroup A component from virus mixtures of A and B types. Two strains of subgroup A, strain
after virus inoculation and tested in the CF test for gs-1 antigen of FeLV (Sarma et al., 1971a).
A were identified as type A by viral interference tests (Sarma and Log, 1973). Human embryo cultures infected with A + B or A + B + C virus mixtures contained either B virus alone or a virus mixture of A and B. A reexamination of these inoculated human cultures for the antigenic type(s) of progeny virus gave the same results. The FL-237 strain of FeLV (mixture of A + C) failed to cause productive infection of whole human embryo cultures. As described above, purified single antigenic types of A and C derived from this strain similarly failed to infect the human embryo cells (Tables 1 and 3). As shown in Table 3, three FeLV mixtures of A + B viruses were purified into subgroup B virus by the described passage in whole human embryo cultures, whereas a similar passage of two other virus mixtures of A and B did not result in such a removal of the A virus components.
F-6 and strain FL-237 (purified from virus mixture of A + C), failed to infect the human embryo cells and were thus similar to strains CT-7 (Gardner) and MAH (Sarma). On the other hand, two single antigenie types of A virus (strains F-10 and FL-163), as well as antigenic mixtures of A + B and A + B + C viruses used in these studies induced productive infection of whole human embryo cells as determined DISCUSSION of CF antigen induction test (Sarma et al., Heterologous human and dog cells which 1971a). The progeny virus derived from cultures infected with single antigenic type we found to be fully susceptible to strain
FELINE LEUKEMIA-SARCOMACOMPLEX TABLE 3
443
virus. On the other hand, as described, we
REMOVALORFAILURETO REMOVETHE A VIRUSCOMPO-found that F-10 and FL-163 strains of NENT FROM VIRUS MIXTURES OF A AND. B BY subgroup A FeLV are able to cause producPASSAGE THROUGH WHOLE HUMAN EMBRYO tive infection of whole human embryo cells. CULTURES Further characterization of the complete
host range of these strains is presently in progress. Our preliminary studies suggest that certain virus mixtures of A and B types can be purified into subgroup B type by pasA A F-10 sage in whole human embryo cells suggestA A FL-163 ing that the subgroup A component present B F-4 A+B in these virus mixtures may not infect B FL-75 A+B whole human embryo cells. The support for B FL-165 A+B this contention comes from our preliminary ST-FeLV A+B A+B None observation that purified subgroup A FeLV FL-237 A+C FL-74 A+B A+B+C derived from one of these virus mixtures (strain F-4) of A and B viruses indeed does n Typing was done by viral interference tests in not infect human cells. On the other hand, feline embryo cultures (Sarma and Log, 1973). the passage in whole human embryo culST-FeLV of subgroup B and partially or tures of uncloned ST-FeLV (A + B types) completely resistant to two strains of sub- and uncloned FL-74 (A + B + C types) did group A [MAH (Sarma) and CT-7 not result in the elimination of the A (Gardner)], have been used by other inves- component present in these virus mixtures. tigators in the studies of feline sarcoma We believe that the A strains present in virus and their associated leukemia viruses these mixtures are infectious for whole (McAllister et al., 1973; Arnstein, P., per- human embryo cells. This observation will sonal communication, 1971). The original be confirmed with cloned subgroup A virus GA strain of feline sarcoma virus (GA- we are now in the process of preparing from FSV) and the accompanying leukemia these stocks. Thus, our observations sugvirus (GA-FeLV) used in these studies are gest that the recovery of both A and B mixtures of A and B viruses (Sarma et al., types by Jarrett et al. (1973) by passage of 1971b, 1971c). However, we found that their FeLV No. 5 through human cells can cells chronically infected with GA-FeLV, be attributed to the parallel infectivity for such as human rhabdomyosarcoma cells human cells of both A as well as B types RD (obtained from Dr. R. M. McAllister) present in their virus mixture rather than and beagle dog embryo cells (obtained due to entry of A type viral genome into from Dr. P. Arnstein) release only the human cells with B-type envelope (phenosubgroup B FeLV. This initial observation typic mixing) as originally believed (Jarsuggesting a partial or complete resistance rett et al., 1973). of human and dog cells to subgroup A virus We found that FL-237 strain of FeLV of led to the present studies. We found that subgroup C has a host range that is neither dog cells are partially resistant to CT-7 as wide as that of strain ST-FeLV of (Gardner) strain of subgroup A virus and subgroup B virus nor as narrow as that of that whole human cells and cells of many strain FL-237 of subgroup A virus. In different mammalian species fail to sup- repeated studies we found this strain to be port the replication of this strain. Our infectious for only certain, but not all, recent quantitative studies with relatively human cells. The FL-237 strain also large doses of CT-7 (Gardner) strain of showed a peculiar tropism for guinea pig subgroup A FeLV ( lo5 infectious units) has cells not displayed by the viruses of subshown that the insusceptibility of human groups A and B we examined. cells to this strain of subgroup A FeLV We found that in every case where puricannot be overcome with large doses of fied FeLV of subgroups A, B, or C caused Virus strain
Envelope antigenie types present
Envelope antigenic type(s) isolated after passage through human cells”
444
SARMA ET AL.
demonstrable virus infection in a heterologous host species, there was no host range modification of virus, demonstrable by a loss of viral infectivity for the homologous host cells, FEF. In addition, the viral envelope specificity demonstrable by viral interference tests was not modified by passage through such heterologous host cells. Jarrett et al. (1973) found that, of the cells of these three species they studied, FeLV of subgroup A was infectious only for cat cells, whereas FeLV of subgroups B and C infected cat, dog, as well as human cells. As described herein, we found that strain ST-FeLV of subgroup B has the widest host range, being able to cause productive infection of a variety of heterologous host cells including human and dog cells. We found that strain CT-7 (Gardner) of subgroup A strain has the narrowest host range. In addition to cat cells, this strain infected dog cells as well. More importantly, we found two strains (F-10 and FL-163) of subgroup A virus to be infectious for whole human embryo cells. In our hands, FL-237 strain of subgroup C virus, found to be infectious for human cells by Jarrett et al. (1973), failed to cause productive infection of certain normal human cells such as whole human embryo cells and human skin and muscle cells; thus, this virus cannot be considered as infectious for human cells as ST-FeLV strain of subgroup B we have described herein. In these studies we found interesting virus strain specific differences within A subgroup in their ability to infect human cells. One strain of subgroup C we studied revealed intraspecies specific differences within a host species (human). The factor(s) responsible for the differential sensitivities of different strains of FeLV are presently unknown. If viral envelope antigens are responsible for the observed differences, it is not readily apparent why subgroup A viruses show a differential pattern in their ability to cause productive infection of heterologous human cells. Minor envelope antigenic differences within members of a subgroup may exist which may account for observed differences in host range. This remains to
be determined. Further studies with additional strains of subgroup A, B, and C viruses are needed before seriously considering viral envelope antigens as being responsible for the observed differences in the host range of these viruses. While additional strains of subgroup A and B are available for this study, at present there exists only one known strain of subgroup C virus; the FL-237 strain and FL-74 strain of subgroup C were derived from the same source (Theilen, G. H., personal communication). It also remains to be determined whether the observed insusceptibility of certain heterologous host cells to FeLV is due to an intracellular block in virus replication as has been shown for N and B tropic mouse type C viruses in mouse cells of B and N types, respectively (Huang et al., 1973; Krontiris et al., 1973). Although these studies have only considered productive infection as a sign of infection, nonproductive infections of heterologous host cells as occurs when avian sarcoma viruses infect mammalian cells (Huebner et al., 1964) have to be considered. Whereas avian sarcoma viral gs antigens can be found in such mammalian cells (Huebner et al., 1964), we failed to find the gs antigens of FeLV detectable by CF test in heterologous cells we found to be insusceptible to productive infection with FeLV. Hardy et al. (1973) and Jarrett et al. (1973) recently showed that FeLV has a remarkable capacity to undergo horizontal transmission to uninfected cats under natural (Hardy et al., 1973) and experimental (Jarrett et al., 1973) conditions accounting for a significant proportion of the cancers of this domestic pet. Our findings on the host range of FeLV types, especially the wide host range of ST-FeLV strain of subgroup B, may have important implications in the natural spread of these viruses to other species. Our seroepidemiological studies of cats and humans have shown that FeLV of all three subgroups are widespread in domestic cats as evidence by the presence of significant levels of virus-neutralizing antibodies in cats with or without neoplasia (Sarma et al., 197313). However, humans failed to reveal the occurrence of overt
FELINE
LEUKEMIA-SARCOMA
natural infections with FeLV demonstrable by the development of virus-neutralizing serum antibodies in subjects in close contact with cat and/or FeLV, such as veterinarians and laboratory workers engaged in FeLV research. The feline leukemia viruses resemble the avian leukosis viruses in the phenomenon of type-specific viral interference (Steck and Rubin, 1966a, 1966b, and Vogt, 1970) and in their ability or inability to cross species barrier and infect cells of heterologous host species (Hanafusa, 1966). The presently discernible difference between the avian leukosis viruses and the feline leukemia-sarcoma viruses appears to be in the relative susceptibilities of homologous host cells to infection with the different antigenic types of virus; whereas, cat cells are uniformly susceptible to the three serotypes of FeLV we have described, the avian cells show a differential pattern of susceptibility or resistance to viruses of different avian leukosis subgroups (Vogt and Ishizaki, 1965; Crittenden, 1968; Payne et al., 1968; Duff and Vogt, 1969; Vogt, 1970). Further studies with cat cells from “inbred” cats may be necessary to determine whether cells from such cats may show differential susceptibilities to the described serotypes of FeLV. ACKNOWLEDGMENT This work NIH-NOl-CP-43254 of the National 20014.
was supported by Contract within the Virus Cancer Program Cancer Institute, Bethesda, MD REFERENCES
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