Vol. 9, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1979, p. 154-156 0095-1137/79/01-0154/03$02.00/0
Feline Leukemia Virus: Survival Under Home and Laboratory Conditions DONALD P. FRANCIS,* M. ESSEX, AND DAWN GAYZAGIAN Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115 Received for publication 3 November 1978
Feline leukemia virus maintained its infectiousness in cell culture medium at 37°C or lower for at least 48 h. However, virus in saliva or medium was inactivated within a few hours if allowed to dry. Feline leukemia virus (FeLV) infection of cats is a classic example of a horizontally transmitted, oncogenic virus of outbred mammals. Horizontal transmission of FeLV has been well documented by serological comparisons of exposed and unexposed cat populations (2,8-10), by investigations of clusters of cases (1, 8), and by prospectively following infected cat populations (1, 2, 9, 10, 15). In a previous paper, we reported that saliva appeared to be the primary source of FeLV responsible for the community spread of this naturally occurring oncogenic virus. The implications of that finding were: (i) cat-to-cat transmission is primarily via saliva, and (ii) healthy pet cats that excrete FeLV expose humans to high concentrations of a potentially oncogenic agent (6). To our knowledge, the heat stability of this virus in saliva and tissue culture media has not been determined. In this paper, we extend our previous findings and report the stability of this virus once released into the environment. These studies included salivary FeLV survival on a dry surface similar to surfaces found in homes and FeLV survival in laboratory culture medium to simulate conditions in research laboratories. Saliva was collected from a cat whose FeLV titer had remained consistently between 103 and 104 focus-forming units (FFU) per ml for almost a year. After injecting a narcotizing dose of ketamine hydrochloride, we put one drop of atropine on the cat's tongue and collected drooled saliva in an iced petri dish. Samples of saliva were made and kept frozen at -90°C until tested. For survival of FeLV in culture medium, we used standardized supernatant virus collected from a feline lymphoma cell line (F422) that produced FeLV-A in titers of 105 to 106 FFU/ml (14). To test for FeLV survival in saliva, we smeared 0.1 ml of saliva over a 20-mm diameter cover slip and then allowed it to stand at room
temperature (240C and 24% relative humidity) inside a laminar flow biohazard hood. At predetermined times, including zero time, cover slips were removed and placed in centrifuge tubes containing 1.2 ml of culture medium (McCoy 5A with 15% fetal bovine serum, penicillin 100 IU/ml, streptomycin 100 ug/ml, and amphotericin, 0.25 ,ug/ml). After being mixed, the specimens were kept frozen at -90°C until tested. To test for virus survival in cell culture medium, we made dilutions of our stock cell-free F422 supernatant in closed plastic tubes (NUNC+, Vanguard International, Red Bank, N.J.), and these tubes were subjected to various temperatures (4, 24, 37, and 5600). At zero time and subsequent determined times, the tubes were removed and frozen at -90°C until tested. Titers of infectious FeLV were determined by a modified method of Fischinger et al. (5, 6). Briefly, triplicate samples were diluted appropriately in cell culture medium (as described above) and placed over day-old, diethylaminoethyl dextran-treated CCC81 monolayers in sixwell cluster plates (Falcon Plastics, Oxnard, Calif.) Foci were read directly at day 13, and titers were reported in FFU. Titers of infectious salivary FeLV dropped rapidly in saliva which was allowed to dry on glass cover slips at room temperature. Saliva originally containing 103 to 104 FFU per ml dropped to 102 to 103 FFU before zero time specimens could be taken and, within 3 h, contained fewer than 10 FFU/ml (Fig. 1). Identical loss of infectious virus was observed for FeLV from a nonsaliva source of virus (F422 supernatant) when it was allowed to dry (data not shown). Levels of infectious FeLV in media dropped to fewer than 10 infectious units within 60 min after the drying occurred. In contrast to the rapid decay of infectiousness observed for dry virus, cell-free FeLV in cell culture medium appeared quite stable. Except for an initial decline in titer before the collection of the first temperature-exposed sample, there 154
VOL. 9, 1979
103 .
i
102
in
2 10I
10I
155
NOTES
I
180 240 120 HMIUTES FIG. 1. FeLVs urvwal i salia allowed to dry on glass cover slips at room temperature (240C). Each dot represents the mean of triplicate portions from a 0
single sample.
60
food and water containers. In addition to FeLV transmission to other cats, owners of infected pet cats may be exposed to high levels of FeLV depending on their handling characteristics and social interactions with cats. In the laboratory, the potential for human infection seems to be considerable. Virus survival results presented here for FeLV are similar to those reported for murine retroviruses (12) and could be typical for retroviruses as a whole. Unlike the oncogenic murine retroviruses, however, the FeLVs grow readily in cultured human cells (4). The stability of FeLV in laboratory media at room temperature and at 370C makes it necessary to protect humans from direct contact with virus-containing fluids in the laboratory. As stipulated by National Institutes of Health guidelines (1), laboratory workers must be protected by the use of biohazar hoods from inhalng aerosols of culture fluids, and al discarded labware should be soaked in virocidal solutions and autoclaved before being removed from the laboratory. The need for these precautions is predicated on the theoretical risk posedby FeLV for humans. The extent, if any, of that risk has yet to be established. Although one recent report identified antibody to FeLV reverse transcriptase on the surface of cells from chronic myelogenous leukemia patients (16), more extensive epidemiological studies are necessary before accurate recommendations based on potential risk of FeLV for humans can be made (3). Two other reports failed to find free serum antibodies to different FeLV proteins in numerous human samples (7, 11). All that we say at this point is that FeLV is present and potentially hazardous
was little loss of infectious virus at 40C up to 48 h. There was sllightly more loss at 22 and at 370C, but survivral was still prolonged. At 56°C, however, there was a rapid loss of infectiousness-dropping below detectable levels within 3 to 4 h (Fig. 2). Transmission of FeLV from an excretor cat to other cats proba bly occurs through close direct contact between cats presumably during face-toface contact. Twro lines of evidence suggest that minimal transmiission of FeLV occurs via aerosols. First, FeLV excretor cats are asymptomatic. Unless they are infected with a secondary respiratory agent, t;hey do not manifest the coughmg and sneezmgr necessary to produce aerosols. Second, there is evidence that uninfected cats caged apart in the same room with FeLV excre100% tor cats do not become infected (9). Conversely, when cats are allowed to interact freely while 40 c residing in the same room essentially all develop serological evidence of infection within 5 months | 108N + 22°C (2, 9, 15). In this report, we document the potential for + 37°C transfer of infectious FeLV from the saliva of an 0 1% excretor cat to a recipient via environmental 4 56°C surfaces. Although the infectiousness of salivary FeLV declines relatively rapidly on dry surfaces, the 30- to 60-min interval during which significant amounts of FeLV can still be detected may be adequate to transfer the agent. This might occur, for example, when several cats are eating from the same dish. The habitual licking charH(XORS acteristic of cats probably represents the primary transmission mechanism, but the relative FIG. 2. FeLV survival in cell culture medium at 4, stability of FeLV suggests that transmission 22, 37, and 56°C. Each dot represents the mean of could also occur through the sharing of common three aliquots of the same sample. *
h.
156
NOTES
to humans who either work in FeLV research laboratories or own FeLV-excreting pet cats. This work was supported by Public Health Service grants CA 13885, CA 18216, and CA 64001 from the National Cancer Institute and by grant PDT-30C from the American Cancer Society. Donald Francis is a Career Development Officer, Bureau of Epidemiology, Center for Disease Control, U.S. Public Health Service, Department of HEW, Atlanta, Ga.
LITERATURE CITED 1. Cotter, S. M., M. Essex, and W. D. Hardy, Jr. 1974. Serological studies of normal and leukemic cats in a multiple-case leukemia cluster. Cancer Res. 34: 1061-1069. 2. Essex, M., S. M. Cotter, A. H. Sliski, W. D. Hardy, Jr., J. R. Stephenson, S. A. Aaronson, and 0. Jarrett. 1977. Horizontal transmission of feline leuke3. 4.
5.
6.
7.
mia virus under natural conditions in a feline leukemia cluster household. Int. J. Cancer 19:90-96. Essex, M., and D. P. Francis. 1976. The risk to humans from malignant diseases of their pets: an unsettled issue. J. Am. Anim. Hosp. Assoc. 12:386-390. Essex, M., G. Klein, F. Deinhardt, L. Wolfe, W. D. Hardy, Jr., G. Theilen, and L. Pearson. 1972. Induction of the feline oncornavirus-association cell membrane antigen in human cells. Nature (London) 238: 187-189. Fischinger, P., C. Blevins, and S. Nomura. 1974. A simple, quantitative assay for both xenotropic murine leukemia and ecotropic feline leukemia viruses. J. Virol. 14: 177-179. Francis, D. P., M. Essex, and W. D. Hardy, Jr. 1977. Excretion of feline leukemia virus by naturally infected pet cats. Nature (London) 269:252-254. Gardner, M. B., J. C. Brown, H. P. Charman, J. R. Stephenson, R. W. Rongey, D. E. Hauser, F. Diegmann, E. Howard, R. Dworsky, R. V. Gilden, and R. J. Huebner. 1977. Feline leukemia virus epidemiol-
J. CLIN. MICROBIOL. 8. 9.
10.
11.
12.
13.
14.
15.
16.
ogy in Los Angeles cats: appraisal of detection methods. Int. J. Cancer 19:581-589. Hardy, W. D., Jr., L. J. Old, P. W. Hess, M. Essex, and S. M. Cotter. 1973. Horizontal transmission of feline leukemia virus. Nature (London) 244:266-269. Hoover, E. A., R. G. Olsen, W. D. Hardy, Jr., and J. P. Schaller. 1977. Horizontal transmission of feline leukemia virus under experimental conditions. J. Nat. Cancer Inst. 58:443-444. Jarrett, W., 0. Jarrett, L. Mackey, H. Laird, W. D. Hardy, Jr., and M. Essex. 1973. Horizontal transmission of leukemia virus and leukemia in the cat. J. Nat. Cancer Inst. 51:833-841. Krakower, J. N., and S. A. Aaronson. 1978. Seroepidemiologic assessment of feline leukemia virus infection risk for man. Nature (London) 273:463-464. Larson, E. W., G. J. Spahn, P. L. Peters, and R. J. Huebner. 1970. Investigations of survival properties of air-borne murine leukemia virus. J. Nat. Cancer Inst. 44:937-941. National Cancer Institute. 1974. Biologic safety manual for research involving oncogenic viruses. National Cancer Institute, Bethesda, Md. Noronha, F., E. Dougherty, A. Poco, C. Gries, J. Post, and C. Rickard. 1974. Cytological and serological studies of a feline endogenous C-type virus. Arch. Gesamte Virusforsch. 45:235-248. Pederson, N. C., G. Theilen, M. A. Keane, L. Fairbanks, T. Mason, B. Orser, C. H. Chen, and C. Allison. 1977. Studies of naturally transmitted feline leukemia virus infection. J. Am. Vet. Med. Assoc. 38: 1523-1526. Saxinger, W. C., P. C. Jacquemin, and R. C. Gallo. 1978. Evidence for a specific immunological response against reverse transcriptase as evidenced by natural IgG found on the membrane of leukocytes of patients with leukemia, p. 466-470. In P. Bentvelzen, J. Hilgers, and D. S. Yohn, (ed.), Advances in comparative leukemia research. Elsevier/North Holland Biomed. Press, New York.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1979, p. 154-156 0095-1137/79/01-0154/03$02.00/0
Feline Leukemia Virus: Survival Under Home and Laboratory Conditions DONALD P. FRANCIS,* M. ESSEX, AND DAWN GAYZAGIAN Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115 Received for publication 3 November 1978
Feline leukemia virus maintained its infectiousness in cell culture medium at 37°C or lower for at least 48 h. However, virus in saliva or medium was inactivated within a few hours if allowed to dry. Feline leukemia virus (FeLV) infection of cats is a classic example of a horizontally transmitted, oncogenic virus of outbred mammals. Horizontal transmission of FeLV has been well documented by serological comparisons of exposed and unexposed cat populations (2,8-10), by investigations of clusters of cases (1, 8), and by prospectively following infected cat populations (1, 2, 9, 10, 15). In a previous paper, we reported that saliva appeared to be the primary source of FeLV responsible for the community spread of this naturally occurring oncogenic virus. The implications of that finding were: (i) cat-to-cat transmission is primarily via saliva, and (ii) healthy pet cats that excrete FeLV expose humans to high concentrations of a potentially oncogenic agent (6). To our knowledge, the heat stability of this virus in saliva and tissue culture media has not been determined. In this paper, we extend our previous findings and report the stability of this virus once released into the environment. These studies included salivary FeLV survival on a dry surface similar to surfaces found in homes and FeLV survival in laboratory culture medium to simulate conditions in research laboratories. Saliva was collected from a cat whose FeLV titer had remained consistently between 103 and 104 focus-forming units (FFU) per ml for almost a year. After injecting a narcotizing dose of ketamine hydrochloride, we put one drop of atropine on the cat's tongue and collected drooled saliva in an iced petri dish. Samples of saliva were made and kept frozen at -90°C until tested. For survival of FeLV in culture medium, we used standardized supernatant virus collected from a feline lymphoma cell line (F422) that produced FeLV-A in titers of 105 to 106 FFU/ml (14). To test for FeLV survival in saliva, we smeared 0.1 ml of saliva over a 20-mm diameter cover slip and then allowed it to stand at room
temperature (240C and 24% relative humidity) inside a laminar flow biohazard hood. At predetermined times, including zero time, cover slips were removed and placed in centrifuge tubes containing 1.2 ml of culture medium (McCoy 5A with 15% fetal bovine serum, penicillin 100 IU/ml, streptomycin 100 ug/ml, and amphotericin, 0.25 ,ug/ml). After being mixed, the specimens were kept frozen at -90°C until tested. To test for virus survival in cell culture medium, we made dilutions of our stock cell-free F422 supernatant in closed plastic tubes (NUNC+, Vanguard International, Red Bank, N.J.), and these tubes were subjected to various temperatures (4, 24, 37, and 5600). At zero time and subsequent determined times, the tubes were removed and frozen at -90°C until tested. Titers of infectious FeLV were determined by a modified method of Fischinger et al. (5, 6). Briefly, triplicate samples were diluted appropriately in cell culture medium (as described above) and placed over day-old, diethylaminoethyl dextran-treated CCC81 monolayers in sixwell cluster plates (Falcon Plastics, Oxnard, Calif.) Foci were read directly at day 13, and titers were reported in FFU. Titers of infectious salivary FeLV dropped rapidly in saliva which was allowed to dry on glass cover slips at room temperature. Saliva originally containing 103 to 104 FFU per ml dropped to 102 to 103 FFU before zero time specimens could be taken and, within 3 h, contained fewer than 10 FFU/ml (Fig. 1). Identical loss of infectious virus was observed for FeLV from a nonsaliva source of virus (F422 supernatant) when it was allowed to dry (data not shown). Levels of infectious FeLV in media dropped to fewer than 10 infectious units within 60 min after the drying occurred. In contrast to the rapid decay of infectiousness observed for dry virus, cell-free FeLV in cell culture medium appeared quite stable. Except for an initial decline in titer before the collection of the first temperature-exposed sample, there 154
VOL. 9, 1979
103 .
i
102
in
2 10I
10I
155
NOTES
I
180 240 120 HMIUTES FIG. 1. FeLVs urvwal i salia allowed to dry on glass cover slips at room temperature (240C). Each dot represents the mean of triplicate portions from a 0
single sample.
60
food and water containers. In addition to FeLV transmission to other cats, owners of infected pet cats may be exposed to high levels of FeLV depending on their handling characteristics and social interactions with cats. In the laboratory, the potential for human infection seems to be considerable. Virus survival results presented here for FeLV are similar to those reported for murine retroviruses (12) and could be typical for retroviruses as a whole. Unlike the oncogenic murine retroviruses, however, the FeLVs grow readily in cultured human cells (4). The stability of FeLV in laboratory media at room temperature and at 370C makes it necessary to protect humans from direct contact with virus-containing fluids in the laboratory. As stipulated by National Institutes of Health guidelines (1), laboratory workers must be protected by the use of biohazar hoods from inhalng aerosols of culture fluids, and al discarded labware should be soaked in virocidal solutions and autoclaved before being removed from the laboratory. The need for these precautions is predicated on the theoretical risk posedby FeLV for humans. The extent, if any, of that risk has yet to be established. Although one recent report identified antibody to FeLV reverse transcriptase on the surface of cells from chronic myelogenous leukemia patients (16), more extensive epidemiological studies are necessary before accurate recommendations based on potential risk of FeLV for humans can be made (3). Two other reports failed to find free serum antibodies to different FeLV proteins in numerous human samples (7, 11). All that we say at this point is that FeLV is present and potentially hazardous
was little loss of infectious virus at 40C up to 48 h. There was sllightly more loss at 22 and at 370C, but survivral was still prolonged. At 56°C, however, there was a rapid loss of infectiousness-dropping below detectable levels within 3 to 4 h (Fig. 2). Transmission of FeLV from an excretor cat to other cats proba bly occurs through close direct contact between cats presumably during face-toface contact. Twro lines of evidence suggest that minimal transmiission of FeLV occurs via aerosols. First, FeLV excretor cats are asymptomatic. Unless they are infected with a secondary respiratory agent, t;hey do not manifest the coughmg and sneezmgr necessary to produce aerosols. Second, there is evidence that uninfected cats caged apart in the same room with FeLV excre100% tor cats do not become infected (9). Conversely, when cats are allowed to interact freely while 40 c residing in the same room essentially all develop serological evidence of infection within 5 months | 108N + 22°C (2, 9, 15). In this report, we document the potential for + 37°C transfer of infectious FeLV from the saliva of an 0 1% excretor cat to a recipient via environmental 4 56°C surfaces. Although the infectiousness of salivary FeLV declines relatively rapidly on dry surfaces, the 30- to 60-min interval during which significant amounts of FeLV can still be detected may be adequate to transfer the agent. This might occur, for example, when several cats are eating from the same dish. The habitual licking charH(XORS acteristic of cats probably represents the primary transmission mechanism, but the relative FIG. 2. FeLV survival in cell culture medium at 4, stability of FeLV suggests that transmission 22, 37, and 56°C. Each dot represents the mean of could also occur through the sharing of common three aliquots of the same sample. *
h.
156
NOTES
to humans who either work in FeLV research laboratories or own FeLV-excreting pet cats. This work was supported by Public Health Service grants CA 13885, CA 18216, and CA 64001 from the National Cancer Institute and by grant PDT-30C from the American Cancer Society. Donald Francis is a Career Development Officer, Bureau of Epidemiology, Center for Disease Control, U.S. Public Health Service, Department of HEW, Atlanta, Ga.
LITERATURE CITED 1. Cotter, S. M., M. Essex, and W. D. Hardy, Jr. 1974. Serological studies of normal and leukemic cats in a multiple-case leukemia cluster. Cancer Res. 34: 1061-1069. 2. Essex, M., S. M. Cotter, A. H. Sliski, W. D. Hardy, Jr., J. R. Stephenson, S. A. Aaronson, and 0. Jarrett. 1977. Horizontal transmission of feline leuke3. 4.
5.
6.
7.
mia virus under natural conditions in a feline leukemia cluster household. Int. J. Cancer 19:90-96. Essex, M., and D. P. Francis. 1976. The risk to humans from malignant diseases of their pets: an unsettled issue. J. Am. Anim. Hosp. Assoc. 12:386-390. Essex, M., G. Klein, F. Deinhardt, L. Wolfe, W. D. Hardy, Jr., G. Theilen, and L. Pearson. 1972. Induction of the feline oncornavirus-association cell membrane antigen in human cells. Nature (London) 238: 187-189. Fischinger, P., C. Blevins, and S. Nomura. 1974. A simple, quantitative assay for both xenotropic murine leukemia and ecotropic feline leukemia viruses. J. Virol. 14: 177-179. Francis, D. P., M. Essex, and W. D. Hardy, Jr. 1977. Excretion of feline leukemia virus by naturally infected pet cats. Nature (London) 269:252-254. Gardner, M. B., J. C. Brown, H. P. Charman, J. R. Stephenson, R. W. Rongey, D. E. Hauser, F. Diegmann, E. Howard, R. Dworsky, R. V. Gilden, and R. J. Huebner. 1977. Feline leukemia virus epidemiol-
J. CLIN. MICROBIOL. 8. 9.
10.
11.
12.
13.
14.
15.
16.
ogy in Los Angeles cats: appraisal of detection methods. Int. J. Cancer 19:581-589. Hardy, W. D., Jr., L. J. Old, P. W. Hess, M. Essex, and S. M. Cotter. 1973. Horizontal transmission of feline leukemia virus. Nature (London) 244:266-269. Hoover, E. A., R. G. Olsen, W. D. Hardy, Jr., and J. P. Schaller. 1977. Horizontal transmission of feline leukemia virus under experimental conditions. J. Nat. Cancer Inst. 58:443-444. Jarrett, W., 0. Jarrett, L. Mackey, H. Laird, W. D. Hardy, Jr., and M. Essex. 1973. Horizontal transmission of leukemia virus and leukemia in the cat. J. Nat. Cancer Inst. 51:833-841. Krakower, J. N., and S. A. Aaronson. 1978. Seroepidemiologic assessment of feline leukemia virus infection risk for man. Nature (London) 273:463-464. Larson, E. W., G. J. Spahn, P. L. Peters, and R. J. Huebner. 1970. Investigations of survival properties of air-borne murine leukemia virus. J. Nat. Cancer Inst. 44:937-941. National Cancer Institute. 1974. Biologic safety manual for research involving oncogenic viruses. National Cancer Institute, Bethesda, Md. Noronha, F., E. Dougherty, A. Poco, C. Gries, J. Post, and C. Rickard. 1974. Cytological and serological studies of a feline endogenous C-type virus. Arch. Gesamte Virusforsch. 45:235-248. Pederson, N. C., G. Theilen, M. A. Keane, L. Fairbanks, T. Mason, B. Orser, C. H. Chen, and C. Allison. 1977. Studies of naturally transmitted feline leukemia virus infection. J. Am. Vet. Med. Assoc. 38: 1523-1526. Saxinger, W. C., P. C. Jacquemin, and R. C. Gallo. 1978. Evidence for a specific immunological response against reverse transcriptase as evidenced by natural IgG found on the membrane of leukocytes of patients with leukemia, p. 466-470. In P. Bentvelzen, J. Hilgers, and D. S. Yohn, (ed.), Advances in comparative leukemia research. Elsevier/North Holland Biomed. Press, New York.
