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A latent virus of rats isolated in tissue culture
VIROLOGY
7,428-437
A Latent
(1959)
Virus L. National
of Rats KILHAM Institutes Accepted
Isolated AND
in Tissue
L. J.
Culture’
OLIVIER~
of Health,
Bethesda,
January
9, 1959
Maryland
Several strains of a new virus (RV) have been isolated from rat tumors under conditions which suggest that it may be a latent agent in t,hese animals. The virus is characterized by its ability to multiply and to cause a marked cytopathogenic effect (CPE) in rat embryo cells in tissue culture. The virus is associated with a hemagglutinin for guinea pig red cells, and circulating antibodies against these strains have been found in a number of laboratory and wild rats, suggesting that it is widely disseminated. Viral antibodies were detected in a germ-free rat, suggesting that the agent may be vertically transmitted. The virus resembles SE polyoma virus in a number of respects, including heat stability, resistance to ether, agglutination of red cells without spontaneous elution and the destruction of red cell receptors by cholera vibrio filtrates containing receptor-destroying enzyme (RDE). The virus differs from SE polyoma virus in that there is no detectable antigenic relationship and the agent does not grow in mouse embryo tissue nor does it cause tumors or other detectable pathology on inoculation into rats. However, the resemblances suggest that the two agents may represent mouse and rat variants of a similar class of virus agent. INTRODUCTIOS
The present study originated in an attempt to apply the methods used by Stewart et al. (1957) in isolating a tumor-inducing virus from mice to the search for a similar agent in rats. Their methods have been further described in other publications (Eddy et al., 1958 a, b, c) . An apparently new agent, the rat virus (RV), has been found and results given below suggest that it has interest from a number of points of view. 1 From the United States Department of Health, Education, Public Health Service, National Institutes of Health, Division Standards (Laboratory of Bacterial Products), Bethesda, Maryland. 2 National Institute of Allergy and Infectious Diseases (Laboratory Diseases). 428
and Welfare, of Biologics of Tropical
9
LATEiYT
VIRW5
OF
RATS
MATERIALS
ISOLATED
AND
IN
TISSUE
CULTURE
429
METHODS
Tissue Cdtures Intact uteri containing embryos were removed from random-bred Sprague-Dawley rats, estimated to be midway through the gestation period. The embryos were separated from uterine and placental tissues, washed in six changes of Hanks’ balanced salt solution containing 10% horse serum, and then minced with scissors. The resulting brei was washed two more times before suspending in a solution of 0.25 % trypsin. Following trypsinization and straining, the cells were given two additional washings, this time in medium 199 containing 2 % calf serum, and the medium was used for the 1:200 suspensions of cells placed in Blake bottles. The fluid was changed in 1 or 2 days, using one-half the amount of calf serum but twice the amount of bicarbonate (1.25 g per liter). The cell sheet was retrypsinized within 5 to 7 days and cells from one Blake botble were used to implant’ 15 to 20, 2-ounce prescription bottles. These final embryo tissue cultures were maintained in medium 199 wit’h 1% calf serum and were ready to be inoculated wit’hin 2 days. Nutrient fluids were changed once a week, and all media contained penicillin and streptomycin. Hemagglutination
(HA)
In testing for hemagglutination titers, supernatant fluids from infected tissue cultures mere carried through serial two-fold dilutions in volumes of 0.5 ml, using buffered saline as a diluent. Equal volumes of 0.5% guinea pig erythrocytes mere then added to each tube. The cells settled at room temperature and results could be read by the pattern method after an hour. In hemagglutination-inhibition (HI) tests, serum dilutions, beginning at 1: 20, were incubat’ed wit’h 8 HA units of virus for half an hour before adding 0.5 % red cells. The volumes of serum dilut,ions, virus, and of cells were each 0.2 ml. These test.s were carried out at, room temperature. Neutralization
Tests
Undiluted serum and virus from infected tissue fluids diluted 1: 3 were mixed in equal volumes of 0.5 ml each, incubated for 15 minutes at room temperature, then added to the cells of a single 2-ounce bottle. This brought the total fluid volume to 6 ml. Results were apparent within 2 weeks. Typical cytopathogenic effect combined with the presence of hemagglutinins in supernatant fluids appeared in the presence of normal,
430
KILHAM
AND
OLIVIER
but not in the presence of specific, immune serum, prepared in guinea pigs. Virus might appear in each bottle if tests were kept 4 weeks or more. Readings were t’herefore made at an earlier period determined in relation to results in control bottles. Sera used in these and in the HI tests were inactivated by heating at 56” for $4 hour. Isolation
and Passage of Virus
Rat tissues selected for virus isolation were minced in a 1: IO suspension with medium 199 containing 2 % calf serum, then added to the cultures of rat embryo in volumes of 0.5 ml and of I .O ml. Supernatant fluids were changed within 24 hours. Each culture was maintained for 3 or 4 weeks, after lvhich the cell sheet was scraped off and 2 ml of fluid and cells carried over to fresh tissue cultures for a succeeding passage. Isolations
of Virus
Three isolations of rat virus (RV) were made from minced tissues. Two of these isolations were from Fisher strain rats which had developed metastasizing sarcomas of the liver associated with encysted Cysticercus fasciolaris the larval stage of Taenia taeniaefomis (Dunning and Curtis, 1957). Pieces of tumor, cysts, and larvae were used for these isolations. A third isolation of RV was from a weanling rat, of the Osborne-Mendel strain bearing a transplantable leukemia described by Curtis and Dunning (1957), a mince of tumor and spleen being used as the original inoculum in tissue culture. These three strains appear to be identical serologically. Guinea pig immune sern, prepared against each of two of the strains, neutralized all three isolates in tissue culture neutralization and in HI tests. The fact that the rats had tumors could have been incidental to these isolations. A more immediate question was whether the virus (RV) originated in the rat embryo tissue cultures or from the tissues of older rats implanted into them. Circumstances under which isolat’ions were made indicated that the latter situation was the most probable. Thus, the virus from the Osborne-Mendel rat appeared on first bissue culture passage, from an original inoculum of mixed spleen and tumor. Ko CPE or HA appeared in three to five passages made with other materials started in or carried through the same lot of rat embryo tissue culture. These materials included normal rat embryo, mouse leukemic tumor, deer papilloma, and tumor and spleen from another Fisher rat from which no virus was recovered in five subsequent passages. Uninoculated control bottles showed no CPE. When the second passage of an isolation from the
A
LATEST
VIRUS
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RATS
ISOLATED
IX
TISSUE
CULTCRE
431
Fisher strain rats was repeated at a later date, CPE and HA appeared in the bottles inoculated. This was not true of simultaneous passages of control materials which had been carried in the same lot of rat embryo tissue culture in which the Fisher isolate had started originally. Cross checks, therefore, gave no suggestion that, RV was arising primarily from any of the tissue cultures involved. In an exceptional instance, however, RV was present in one lot of rat embryo tissue culture, apparently from the time of its first preparation. The cells of this lot sheeted out well after retrypsinization, but 4 to 6 days later all 40 bottles of the lot showed a typical CPE, regardless of whether inoculat)ed or not. Unfortunately, the entire lot was discarded by accident, with t,he exception of 2 bottles. Fluids from these 2 had an HA titer of 1: 64 and the virus was transferred in a second passage. This isolation of RV was unexpected and was not fully controlled. There was no way of repeating it from the original source material. Tissue
Culture
Passage
Effects produced in rat embryo tissue cultures depended somewhat on the concent,ration of virus in the original inoculum. Undiluted fluids from infect,ed t’issue cultures usually lead to cytopathogenic effects accompanied by HA titers of 1: 64 to 1: 512 in the supernatant fluids in from 9 to 12 days at 36”. Figure 1 is a photograph of normal and Fig. 2 of RV-infected rat embryo tissue culture cells. The infected cells have formed dense masseswhich have thin extensions radiating outward. Titrations of RV were carried out in tissue culture on a few occasions. An infectivit#y t’iter of 10-j was obtained from one of the viruses isolated, but it was 4 weeks before the virus at this dilution led to HA and a typical CPE. By this time cell sheets were deteriorating in uninoculated bott’les. Such deterioration, however, did not resemble the typical CPE, nor was it ever found associated with HA. The three isolates made of RV have been maintained through four, six, and ten tissue culture passages, respectively. Physical
Properties
Rat virus is filterable and relatively stable. Each of three strains isolated has passedthrough Seitz filters (ST-l), shown to be bacteria-tight by a simultaneous addition of Escherichia coli. Ether resistance was demonstrated by keeping a suspensionof RV in the presence of anesthetic ether’for 18 hours at 4’. This treatment did not affect the ability of the agent to multiply in bissuecult,ure, as compared wit,h an untreated con-
432
KILHAM
FIG.
1. A normal,
AND
uninoculated
OLIVJER
rat
embryo
tissue
culture.
trol. RV is also relatively resistant to heat inactivation. Virus preparations employed in these test’s were submerged in water baths within sealed ampoules. Infectivity was retained in three samples, exposed for $$, 1, and 2 hours, respectively, at 80”, although no hemagglutination took place when these samples were tested for HA immediately after exposure. RV withstands storage for six months or longer at -40”. Table 1 presents phenomena associated with RV hemagglutination. The virus hemagglutinates but does not elute appreciably from guinea pig erythrocytes. This was true whether the HA tests were performed at de23’ (room temperature), 36”, or 40”. A potent RDE preparation stroyed its erythrocyte receptors. Hemagglutinatin-adsorption does not
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VIRUS
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R9TS
ISOLATED
In-
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FIG. 2. Rat embryo tissue culture infected with rat virus (RV) showing cytopathogenic effect. Clumping and rounding of cells, long extensions from such masses, and a widespread thinning of the cell sheet are characteristic. Hematoxylin and eosin; magnification: X 330.
take place when guinea pig eryt’hrocytes are added to RV-infected tissue culture. No hemagglutination has been observed on addition of human “O”, human cord, or chicken erythrocytes to suspensions of rat virus. There is agglutination of rat erythrocytes, but results are less easily read than with guinea pig cells. Inoculation
of Various Animals
Rat virus produces no apparent illness in a variety of laboratory animals tested. These included suckling hamsters, suckling mice of Swiss
434
KILHAM
AND
OLIVIER
TABLE COMPARISON OF RAT AND GENERAL PHYSICAL
1
SE POLYOMA VIRUSES IN REGARD AND BIOLOGICAL PROPERTIES Rat Virus
Hemagglutination (G.P.RBC) Elution from (G.P. RBC) Tissue culture hemadsorption (G.P. RBC) Effect RDE on G.P. RBC Exposure to ether Effect of heat on infectivity Mouse embryo tissue culture Rat embryo tissue culture SE Polyoma immune G.P. serum Rat virus immune G.P. serum a Guinea b Brodsky,
pig. I., Rowe,
Room
temp.
None None
at room
TO
SE Polyoma
4O c temp.
At room None
temp.
Destroys virus receptors Resistant Diminished, not lost x-2 hr 80” C Xo growth
Destorys virus receptors Resistant Diminished, not lost 65-70” C b CPE and HA
CPE and HA Neut. and HI neg. Neut. and HI pos
No growth Neut. and pos. Neut. and neg.
W. P., Hartley,
tests tests
J. W., and
Lane,
HI
tests
HI
tests
W. (in preparation).
albino and CSH strains, and newborn rats of the Sprague-Dawley strain, inoculated subcutaneously when less than 24 hours old; meanling Swiss mice and weanling rats of the Sprague-Dawley and of several inbred strains which included the JU/CN, JUG/CN, and the Lobund strains, inoculated intraperitoneally and intracerebrally with RV. Ko illness appeared in rabbits or guinea pigs immunized with RV, given intravenously or intraperitoneally. No hemagglutinins appeared in three amniotic passages of RV in embryonated eggs. None of the mice, rats, or hamsters kept for from three to six months have developed tumors. Distribution
of Naturally
Occurring
Antibodies
Table 2 gives results of hemagglutination inhibition (HI) and of tissue culture neutralization tests performed with rat virus (RV). Results by both tests have been in agreement. Thus, sera from one mouse, two rabbits, and three normal guinea pigs had no demonstrable antibodies by either test. This was also true of one normal rat of the Fisher strain. A group of eight other rats had antibodies by both tests and included three
A
LATEKT
VIRUS
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RATS
ISOLATED
TABLE RESULTS
IN
TISSUE
435
CULTURE
2
OF TISSUE CULTURE (TC) NEUTRALIZATION AND OF INHIBITION HEMAGGLUTINATION (HI) TESTS IN SERA OF VARIOUS ANIMALS RUN AGAINST RAT VIRUS (RV) Animal
Remarks
Normal Normal Normal Sormal Xormal Normal Normal Xormal Sarcomac Normal Normal Normal Immunized Immunized
Mouse 3 Guinea pigs 2 Rabbits Fisher rat A Fisher rat B Fisher rat C Fisher rat D Fisher rat E Fisher rat Sp.-Ijaw.* rat A Sp.-Ijaw. rat B Sp.-Daw. rat C Guinea pig 1 Guinea pig 2 a No inhibition at 1:20. 8 Sprague-Dawley strain. c Associated with Cysticercus
TC Nat.
xeg. Neg. Neg.
HI Titer
POS.
None” None None 1:160 1:320 1:40
Neg. Pos Pos . Pos. Pos. 1’0s. Pos. Pos.
1:80 1:20 1: 160 1:80 1:80 1:80 I:160
POS.
1’0s.
vs. RV vs. RV
Test
OF
NOM?
fasiolaris.
rats of t,he Sprague-Dawley and five of the Fisher strain. One of the latter group had a Cysticercus sarcoma. It was evident from these tests that, RV may be widely distributed among laboratory rats. An additional question was whether RV might occur in wild rats (Rattus norvegicus). Eight of these animals, kindly supplied by Dr. J. C. Calhoun of the Kationnl Institute of Mental Health, all had HI titers of from 1: 80 to 1: 640--even higher levels than those obtained from the laboratory strains. A single serum specimen was obtained from a Lobund strain rat at the time of its removal from a germ-free tank.” This animal was over a year old. Its serum had an HI tit)er of 1: 320.
Comparison
of RV to Other Viruses
Encephalomyocarditis (EMC) and the Novy rat virus (Novy et al., 1.953) are the only other viruses known to us which cause spontaneous infection of rat,s, each of them causing paralyses and fatal encephalit,is on inoculation of rats and mice (Kilham et al., 1955; Jordan et al., 1953). 3 Contributed by Met,abolic IXseases.
Mr.
E. G.
Mcljaniel,
Nat.ional
Institute
of Arthritis
anti
436
KILHAM
AND
OLIVIEIZ
Although RV is not pathogenic for these animals, a question was whether it might not be an avirulent form of a previously known agent. Two types of tests were performed in weanling mice in a search for crossimmunity. In one, RV-immune serum was incubated with EMC and Novy virus respectively, then inoculated into mice intraperitoneally, and in the other type of test, mice inoculated with RV in doses of 0.5 ml of infected tissue culture fluid, were challenged a week later with the other two agents. All animals died of illness characteristic of infection with either the EMC or Novy virus. There was thus no demonstrable active or passive protection from RV against either of them. EMC rabbit immune serum had no neutralizing effect on the growth of RV in rat embryo tissue culture. DISCUSSION
St,ewart et al. (1957) used mouse embryo tissue cultures to isolate polyoma virus from mouse parotid tumors, and their success suggested that similar methods might be applied to other animal species. With this in mind several types of rat tumors and associated tissues were inoculated into rat embryo tissue cultures. A new virus was isolated in from one to three passages on three occasions. It resembled polyoma virus in producing an almost identical type of CPE and in being agglutinable to high titer wit,h guinea pig erythrocytes. These similarities, summarized in Table 1, were borne out by further tests. For example, the agent was resistant to et’her, its red cell receptors were destroyed by RDE, and there was no hemadsorption. Certain differences between the two agents are also readily apparent. No relationships have been revealed by serologic tests, and RV has produced neither illness nor tumors in inoculated animals. It should be kept in mind, however, that no suckling rats have been available for inoculation except random-bred Sprague-Dawleys, and rats of this strain have naturally occurring antibodies to RV. It is hoped to investigate the relationships of RV to polyoma in greater detail in continuing studies, to try suckling rats of nonimmune, inbred strains, and to find out, if possible, whether RV may be vertically transmitted, from mother to fetus, as was suggested by one unplanned experiment. Germ-free rats, should more become available, might offer one approach to the latter problem. One of them, as detailed above, has antibodies against RV. ACKNOWLEDGMENT The valuable
senior author wishes technical assistance.
to express appreciation
to Mr.
Leroy
Ssmuels
for
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437
REFERENCES BRODSKY, I., ROWE, W. P., HARTLEY, J. W., AND LANE, W. In preparation. BULLOCK, F. I)., and CURTIS, M. R. (1920). The experimental production of sarcoma of the liver of rats. Pro?. N. Y. Pathol. Sot. 20, 149-175. DUNNING, W. F., and CURTIS, M. R. (1957). A transplantable acute leukemia in an inbred line of rats. J. Natl. Cancer Inst. 19, 845-852. EDDY, B. E., ROWE, W. P., HARTLEY, J. W., STEWART, S. E., and HUEBNER, It. J. (1958a). Hemagglutination with SE Polyoma Virus. Virology 6, 29(t291. EDDY, B. E., STEWART, S. E., and BERKELEY, W. (1958b). Cytopathogenicitp in tissue cultures by a tumor virus from mice. Proc. Sot. Ezptl. Biol. Med. 98, 848-851. EDDY, B. E., STEWART, S. E., YOUNG, R., and MIDER, G. B. (1958c). Neoplasms in hamsters induced by mouse tumor agent in tissue culture. J. N&Z. Cunrer Inst. 20, 747-756. JORIIAIV, R. T., NUNGESTER, W. J., and PRESTON, W. S. (1953). Recovery of the Novy rat virus. 1. Infectious Diseases 93, 124-129. KILHAM, L., MASON, P., and DAVIES, J. N. P. (1955). Pathogenesis of fatal encephalomyocarditis (EMC) virus infections in albino rats. Proc. Sot. Erptl. Biol. Med. 90, 383-387. Novu, F. G., PERKINS, 1%‘. A., CHAMBERS, R., and IIEKRUIF, P. H. (1953). The rat. virus. J. lnfectiozcs Diseases 93, 111-123. STEWART, S. E., EDDY, B. E., GOCHENOUR, A. M., BORGESE, N. G., and GRUBBS, G. E. (1957). The induction of neoplasms with a substance released from mouse tumors by tissue culture. Viroloqq 3, 38&400.
7,428-437
A Latent
(1959)
Virus L. National
of Rats KILHAM Institutes Accepted
Isolated AND
in Tissue
L. J.
Culture’
OLIVIER~
of Health,
Bethesda,
January
9, 1959
Maryland
Several strains of a new virus (RV) have been isolated from rat tumors under conditions which suggest that it may be a latent agent in t,hese animals. The virus is characterized by its ability to multiply and to cause a marked cytopathogenic effect (CPE) in rat embryo cells in tissue culture. The virus is associated with a hemagglutinin for guinea pig red cells, and circulating antibodies against these strains have been found in a number of laboratory and wild rats, suggesting that it is widely disseminated. Viral antibodies were detected in a germ-free rat, suggesting that the agent may be vertically transmitted. The virus resembles SE polyoma virus in a number of respects, including heat stability, resistance to ether, agglutination of red cells without spontaneous elution and the destruction of red cell receptors by cholera vibrio filtrates containing receptor-destroying enzyme (RDE). The virus differs from SE polyoma virus in that there is no detectable antigenic relationship and the agent does not grow in mouse embryo tissue nor does it cause tumors or other detectable pathology on inoculation into rats. However, the resemblances suggest that the two agents may represent mouse and rat variants of a similar class of virus agent. INTRODUCTIOS
The present study originated in an attempt to apply the methods used by Stewart et al. (1957) in isolating a tumor-inducing virus from mice to the search for a similar agent in rats. Their methods have been further described in other publications (Eddy et al., 1958 a, b, c) . An apparently new agent, the rat virus (RV), has been found and results given below suggest that it has interest from a number of points of view. 1 From the United States Department of Health, Education, Public Health Service, National Institutes of Health, Division Standards (Laboratory of Bacterial Products), Bethesda, Maryland. 2 National Institute of Allergy and Infectious Diseases (Laboratory Diseases). 428
and Welfare, of Biologics of Tropical
9
LATEiYT
VIRW5
OF
RATS
MATERIALS
ISOLATED
AND
IN
TISSUE
CULTURE
429
METHODS
Tissue Cdtures Intact uteri containing embryos were removed from random-bred Sprague-Dawley rats, estimated to be midway through the gestation period. The embryos were separated from uterine and placental tissues, washed in six changes of Hanks’ balanced salt solution containing 10% horse serum, and then minced with scissors. The resulting brei was washed two more times before suspending in a solution of 0.25 % trypsin. Following trypsinization and straining, the cells were given two additional washings, this time in medium 199 containing 2 % calf serum, and the medium was used for the 1:200 suspensions of cells placed in Blake bottles. The fluid was changed in 1 or 2 days, using one-half the amount of calf serum but twice the amount of bicarbonate (1.25 g per liter). The cell sheet was retrypsinized within 5 to 7 days and cells from one Blake botble were used to implant’ 15 to 20, 2-ounce prescription bottles. These final embryo tissue cultures were maintained in medium 199 wit’h 1% calf serum and were ready to be inoculated wit’hin 2 days. Nutrient fluids were changed once a week, and all media contained penicillin and streptomycin. Hemagglutination
(HA)
In testing for hemagglutination titers, supernatant fluids from infected tissue cultures mere carried through serial two-fold dilutions in volumes of 0.5 ml, using buffered saline as a diluent. Equal volumes of 0.5% guinea pig erythrocytes mere then added to each tube. The cells settled at room temperature and results could be read by the pattern method after an hour. In hemagglutination-inhibition (HI) tests, serum dilutions, beginning at 1: 20, were incubat’ed wit’h 8 HA units of virus for half an hour before adding 0.5 % red cells. The volumes of serum dilut,ions, virus, and of cells were each 0.2 ml. These test.s were carried out at, room temperature. Neutralization
Tests
Undiluted serum and virus from infected tissue fluids diluted 1: 3 were mixed in equal volumes of 0.5 ml each, incubated for 15 minutes at room temperature, then added to the cells of a single 2-ounce bottle. This brought the total fluid volume to 6 ml. Results were apparent within 2 weeks. Typical cytopathogenic effect combined with the presence of hemagglutinins in supernatant fluids appeared in the presence of normal,
430
KILHAM
AND
OLIVIER
but not in the presence of specific, immune serum, prepared in guinea pigs. Virus might appear in each bottle if tests were kept 4 weeks or more. Readings were t’herefore made at an earlier period determined in relation to results in control bottles. Sera used in these and in the HI tests were inactivated by heating at 56” for $4 hour. Isolation
and Passage of Virus
Rat tissues selected for virus isolation were minced in a 1: IO suspension with medium 199 containing 2 % calf serum, then added to the cultures of rat embryo in volumes of 0.5 ml and of I .O ml. Supernatant fluids were changed within 24 hours. Each culture was maintained for 3 or 4 weeks, after lvhich the cell sheet was scraped off and 2 ml of fluid and cells carried over to fresh tissue cultures for a succeeding passage. Isolations
of Virus
Three isolations of rat virus (RV) were made from minced tissues. Two of these isolations were from Fisher strain rats which had developed metastasizing sarcomas of the liver associated with encysted Cysticercus fasciolaris the larval stage of Taenia taeniaefomis (Dunning and Curtis, 1957). Pieces of tumor, cysts, and larvae were used for these isolations. A third isolation of RV was from a weanling rat, of the Osborne-Mendel strain bearing a transplantable leukemia described by Curtis and Dunning (1957), a mince of tumor and spleen being used as the original inoculum in tissue culture. These three strains appear to be identical serologically. Guinea pig immune sern, prepared against each of two of the strains, neutralized all three isolates in tissue culture neutralization and in HI tests. The fact that the rats had tumors could have been incidental to these isolations. A more immediate question was whether the virus (RV) originated in the rat embryo tissue cultures or from the tissues of older rats implanted into them. Circumstances under which isolat’ions were made indicated that the latter situation was the most probable. Thus, the virus from the Osborne-Mendel rat appeared on first bissue culture passage, from an original inoculum of mixed spleen and tumor. Ko CPE or HA appeared in three to five passages made with other materials started in or carried through the same lot of rat embryo tissue culture. These materials included normal rat embryo, mouse leukemic tumor, deer papilloma, and tumor and spleen from another Fisher rat from which no virus was recovered in five subsequent passages. Uninoculated control bottles showed no CPE. When the second passage of an isolation from the
A
LATEST
VIRUS
OF
RATS
ISOLATED
IX
TISSUE
CULTCRE
431
Fisher strain rats was repeated at a later date, CPE and HA appeared in the bottles inoculated. This was not true of simultaneous passages of control materials which had been carried in the same lot of rat embryo tissue culture in which the Fisher isolate had started originally. Cross checks, therefore, gave no suggestion that, RV was arising primarily from any of the tissue cultures involved. In an exceptional instance, however, RV was present in one lot of rat embryo tissue culture, apparently from the time of its first preparation. The cells of this lot sheeted out well after retrypsinization, but 4 to 6 days later all 40 bottles of the lot showed a typical CPE, regardless of whether inoculat)ed or not. Unfortunately, the entire lot was discarded by accident, with t,he exception of 2 bottles. Fluids from these 2 had an HA titer of 1: 64 and the virus was transferred in a second passage. This isolation of RV was unexpected and was not fully controlled. There was no way of repeating it from the original source material. Tissue
Culture
Passage
Effects produced in rat embryo tissue cultures depended somewhat on the concent,ration of virus in the original inoculum. Undiluted fluids from infect,ed t’issue cultures usually lead to cytopathogenic effects accompanied by HA titers of 1: 64 to 1: 512 in the supernatant fluids in from 9 to 12 days at 36”. Figure 1 is a photograph of normal and Fig. 2 of RV-infected rat embryo tissue culture cells. The infected cells have formed dense masseswhich have thin extensions radiating outward. Titrations of RV were carried out in tissue culture on a few occasions. An infectivit#y t’iter of 10-j was obtained from one of the viruses isolated, but it was 4 weeks before the virus at this dilution led to HA and a typical CPE. By this time cell sheets were deteriorating in uninoculated bott’les. Such deterioration, however, did not resemble the typical CPE, nor was it ever found associated with HA. The three isolates made of RV have been maintained through four, six, and ten tissue culture passages, respectively. Physical
Properties
Rat virus is filterable and relatively stable. Each of three strains isolated has passedthrough Seitz filters (ST-l), shown to be bacteria-tight by a simultaneous addition of Escherichia coli. Ether resistance was demonstrated by keeping a suspensionof RV in the presence of anesthetic ether’for 18 hours at 4’. This treatment did not affect the ability of the agent to multiply in bissuecult,ure, as compared wit,h an untreated con-
432
KILHAM
FIG.
1. A normal,
AND
uninoculated
OLIVJER
rat
embryo
tissue
culture.
trol. RV is also relatively resistant to heat inactivation. Virus preparations employed in these test’s were submerged in water baths within sealed ampoules. Infectivity was retained in three samples, exposed for $$, 1, and 2 hours, respectively, at 80”, although no hemagglutination took place when these samples were tested for HA immediately after exposure. RV withstands storage for six months or longer at -40”. Table 1 presents phenomena associated with RV hemagglutination. The virus hemagglutinates but does not elute appreciably from guinea pig erythrocytes. This was true whether the HA tests were performed at de23’ (room temperature), 36”, or 40”. A potent RDE preparation stroyed its erythrocyte receptors. Hemagglutinatin-adsorption does not
A
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VIRUS
OF
R9TS
ISOLATED
In-
TISSUE
CULTURE
433
FIG. 2. Rat embryo tissue culture infected with rat virus (RV) showing cytopathogenic effect. Clumping and rounding of cells, long extensions from such masses, and a widespread thinning of the cell sheet are characteristic. Hematoxylin and eosin; magnification: X 330.
take place when guinea pig eryt’hrocytes are added to RV-infected tissue culture. No hemagglutination has been observed on addition of human “O”, human cord, or chicken erythrocytes to suspensions of rat virus. There is agglutination of rat erythrocytes, but results are less easily read than with guinea pig cells. Inoculation
of Various Animals
Rat virus produces no apparent illness in a variety of laboratory animals tested. These included suckling hamsters, suckling mice of Swiss
434
KILHAM
AND
OLIVIER
TABLE COMPARISON OF RAT AND GENERAL PHYSICAL
1
SE POLYOMA VIRUSES IN REGARD AND BIOLOGICAL PROPERTIES Rat Virus
Hemagglutination (G.P.RBC) Elution from (G.P. RBC) Tissue culture hemadsorption (G.P. RBC) Effect RDE on G.P. RBC Exposure to ether Effect of heat on infectivity Mouse embryo tissue culture Rat embryo tissue culture SE Polyoma immune G.P. serum Rat virus immune G.P. serum a Guinea b Brodsky,
pig. I., Rowe,
Room
temp.
None None
at room
TO
SE Polyoma
4O c temp.
At room None
temp.
Destroys virus receptors Resistant Diminished, not lost x-2 hr 80” C Xo growth
Destorys virus receptors Resistant Diminished, not lost 65-70” C b CPE and HA
CPE and HA Neut. and HI neg. Neut. and HI pos
No growth Neut. and pos. Neut. and neg.
W. P., Hartley,
tests tests
J. W., and
Lane,
HI
tests
HI
tests
W. (in preparation).
albino and CSH strains, and newborn rats of the Sprague-Dawley strain, inoculated subcutaneously when less than 24 hours old; meanling Swiss mice and weanling rats of the Sprague-Dawley and of several inbred strains which included the JU/CN, JUG/CN, and the Lobund strains, inoculated intraperitoneally and intracerebrally with RV. Ko illness appeared in rabbits or guinea pigs immunized with RV, given intravenously or intraperitoneally. No hemagglutinins appeared in three amniotic passages of RV in embryonated eggs. None of the mice, rats, or hamsters kept for from three to six months have developed tumors. Distribution
of Naturally
Occurring
Antibodies
Table 2 gives results of hemagglutination inhibition (HI) and of tissue culture neutralization tests performed with rat virus (RV). Results by both tests have been in agreement. Thus, sera from one mouse, two rabbits, and three normal guinea pigs had no demonstrable antibodies by either test. This was also true of one normal rat of the Fisher strain. A group of eight other rats had antibodies by both tests and included three
A
LATEKT
VIRUS
OF
RATS
ISOLATED
TABLE RESULTS
IN
TISSUE
435
CULTURE
2
OF TISSUE CULTURE (TC) NEUTRALIZATION AND OF INHIBITION HEMAGGLUTINATION (HI) TESTS IN SERA OF VARIOUS ANIMALS RUN AGAINST RAT VIRUS (RV) Animal
Remarks
Normal Normal Normal Sormal Xormal Normal Normal Xormal Sarcomac Normal Normal Normal Immunized Immunized
Mouse 3 Guinea pigs 2 Rabbits Fisher rat A Fisher rat B Fisher rat C Fisher rat D Fisher rat E Fisher rat Sp.-Ijaw.* rat A Sp.-Ijaw. rat B Sp.-Daw. rat C Guinea pig 1 Guinea pig 2 a No inhibition at 1:20. 8 Sprague-Dawley strain. c Associated with Cysticercus
TC Nat.
xeg. Neg. Neg.
HI Titer
POS.
None” None None 1:160 1:320 1:40
Neg. Pos Pos . Pos. Pos. 1’0s. Pos. Pos.
1:80 1:20 1: 160 1:80 1:80 1:80 I:160
POS.
1’0s.
vs. RV vs. RV
Test
OF
NOM?
fasiolaris.
rats of t,he Sprague-Dawley and five of the Fisher strain. One of the latter group had a Cysticercus sarcoma. It was evident from these tests that, RV may be widely distributed among laboratory rats. An additional question was whether RV might occur in wild rats (Rattus norvegicus). Eight of these animals, kindly supplied by Dr. J. C. Calhoun of the Kationnl Institute of Mental Health, all had HI titers of from 1: 80 to 1: 640--even higher levels than those obtained from the laboratory strains. A single serum specimen was obtained from a Lobund strain rat at the time of its removal from a germ-free tank.” This animal was over a year old. Its serum had an HI tit)er of 1: 320.
Comparison
of RV to Other Viruses
Encephalomyocarditis (EMC) and the Novy rat virus (Novy et al., 1.953) are the only other viruses known to us which cause spontaneous infection of rat,s, each of them causing paralyses and fatal encephalit,is on inoculation of rats and mice (Kilham et al., 1955; Jordan et al., 1953). 3 Contributed by Met,abolic IXseases.
Mr.
E. G.
Mcljaniel,
Nat.ional
Institute
of Arthritis
anti
436
KILHAM
AND
OLIVIEIZ
Although RV is not pathogenic for these animals, a question was whether it might not be an avirulent form of a previously known agent. Two types of tests were performed in weanling mice in a search for crossimmunity. In one, RV-immune serum was incubated with EMC and Novy virus respectively, then inoculated into mice intraperitoneally, and in the other type of test, mice inoculated with RV in doses of 0.5 ml of infected tissue culture fluid, were challenged a week later with the other two agents. All animals died of illness characteristic of infection with either the EMC or Novy virus. There was thus no demonstrable active or passive protection from RV against either of them. EMC rabbit immune serum had no neutralizing effect on the growth of RV in rat embryo tissue culture. DISCUSSION
St,ewart et al. (1957) used mouse embryo tissue cultures to isolate polyoma virus from mouse parotid tumors, and their success suggested that similar methods might be applied to other animal species. With this in mind several types of rat tumors and associated tissues were inoculated into rat embryo tissue cultures. A new virus was isolated in from one to three passages on three occasions. It resembled polyoma virus in producing an almost identical type of CPE and in being agglutinable to high titer wit,h guinea pig erythrocytes. These similarities, summarized in Table 1, were borne out by further tests. For example, the agent was resistant to et’her, its red cell receptors were destroyed by RDE, and there was no hemadsorption. Certain differences between the two agents are also readily apparent. No relationships have been revealed by serologic tests, and RV has produced neither illness nor tumors in inoculated animals. It should be kept in mind, however, that no suckling rats have been available for inoculation except random-bred Sprague-Dawleys, and rats of this strain have naturally occurring antibodies to RV. It is hoped to investigate the relationships of RV to polyoma in greater detail in continuing studies, to try suckling rats of nonimmune, inbred strains, and to find out, if possible, whether RV may be vertically transmitted, from mother to fetus, as was suggested by one unplanned experiment. Germ-free rats, should more become available, might offer one approach to the latter problem. One of them, as detailed above, has antibodies against RV. ACKNOWLEDGMENT The valuable
senior author wishes technical assistance.
to express appreciation
to Mr.
Leroy
Ssmuels
for
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LATENT
VIRUS
OF
RATS
ISOLATED
IN
TISSUE
CULTURE
437
REFERENCES BRODSKY, I., ROWE, W. P., HARTLEY, J. W., AND LANE, W. In preparation. BULLOCK, F. I)., and CURTIS, M. R. (1920). The experimental production of sarcoma of the liver of rats. Pro?. N. Y. Pathol. Sot. 20, 149-175. DUNNING, W. F., and CURTIS, M. R. (1957). A transplantable acute leukemia in an inbred line of rats. J. Natl. Cancer Inst. 19, 845-852. EDDY, B. E., ROWE, W. P., HARTLEY, J. W., STEWART, S. E., and HUEBNER, It. J. (1958a). Hemagglutination with SE Polyoma Virus. Virology 6, 29(t291. EDDY, B. E., STEWART, S. E., and BERKELEY, W. (1958b). Cytopathogenicitp in tissue cultures by a tumor virus from mice. Proc. Sot. Ezptl. Biol. Med. 98, 848-851. EDDY, B. E., STEWART, S. E., YOUNG, R., and MIDER, G. B. (1958c). Neoplasms in hamsters induced by mouse tumor agent in tissue culture. J. N&Z. Cunrer Inst. 20, 747-756. JORIIAIV, R. T., NUNGESTER, W. J., and PRESTON, W. S. (1953). Recovery of the Novy rat virus. 1. Infectious Diseases 93, 124-129. KILHAM, L., MASON, P., and DAVIES, J. N. P. (1955). Pathogenesis of fatal encephalomyocarditis (EMC) virus infections in albino rats. Proc. Sot. Erptl. Biol. Med. 90, 383-387. Novu, F. G., PERKINS, 1%‘. A., CHAMBERS, R., and IIEKRUIF, P. H. (1953). The rat. virus. J. lnfectiozcs Diseases 93, 111-123. STEWART, S. E., EDDY, B. E., GOCHENOUR, A. M., BORGESE, N. G., and GRUBBS, G. E. (1957). The induction of neoplasms with a substance released from mouse tumors by tissue culture. Viroloqq 3, 38&400.