Enhanced Growth of a Murine Coronavirus in Transformed Mouse Cells

Plaque formation by A59 virus, a murine coronavirus, was facilitated in AL/N and Balb mouse cells transformed by polyoma virus, simian virus 40, murine sarcoma virus, or mammary tumor virus. In these virus-transformed cells, A59 virus plaques were larger, they appeared earlier, and plaquing efficiencies were higher than in normal, untransformed cells. “Spontaneously” transformed AL/N cells behaved similarly to untransformed cells, whereas “spontaneously” transformed Balb cells resembled virus-transformed cell hosts. Both untransformed and transformed AL/N and Balb cells were permissive hosts for A59 virus. However, multiplication of A59 virus was enhanced at least fivefold in the virus-transformed AL/N cell hosts. Larger differences (100-fold or greater) in A59 virus production were obtained during the first cycle of infection in Balb cells at low multiplicities and in AL/N cells after multiple cycles of virus growth. In virus-transformed and “spontaneously” transformed Balb cells, A59 virus induced extensive syncytia formation.

Coronaviruses are medium-sized, enveloped, ribonucleic acid viruses which, in negatively stained preparations, appear round and bear a corona of irregular, petal-shaped surface projections (5). Biologically, coronaviruses display marked host specificity. Most of the coronaviruses of man were first identified by the use of human, embryonic tracheal and nasal organ cultures (11,15,20). Several of these viruses have not yet been adapted to a cell culture system (7,8,12). Coronaviruses of avian origin are influenced by phenotypic differences in the avian cell host (13,14) and undergo very limited growth in cell cultures derived fron nonavian species (9,10). Murine coronaviruses, which include mouse hepatitis viruses, have been cultivated only in cells derived from mouse tissues (16). In some cases, the growth of these viruses is markedly affected by the genetic background of the host (6,17).
During an investigation employing transformed cells, we observed pronounced host-dependent differences in the plaquing behavior of several murine coronaviruses.
In this communication the interaction of A59 virus, a murine coronavirus, with normal (untransformed), virus-transformed, and "spontaneously" transformed derivative AL/N and Balb mouse cell lines is documented. (A pre-liminary report of this work was presented at the 70th Annual Meeting of the American Society for Microbiology, Boston, Mass., 26 April to 1 May 1970.) Plaque formation by this virus is shown to be facilitated in virus-transformed and certain "spontaneously" transformed cell types. In these transformed cell hosts, A59 virus yields are increased.
MATERIALS AND METHODS Cell cultures and media. The following prefixes have been employed to identify the origin of each cell type: N, normal (untransformed); ST, "spontaneously" transformed; Py, polyoma virus-transformed; SV, simian virus 40 (SV40) -transformed; MSV, murine sarcoma virus (MSV)-transformed.
Many cell lines of mouse origin were examined as hosts for A59 virus growth, cytopathogenicity, and plaque production. The N-AL/N cell line and its derivatives were developed in this laboratory (19). N-AL/N is a nontumorigenic cell line established from mouse embryos of the AL/N strain. ST-AL/N is a "spontaneously" transformed (tumorigenic) derivative of N-AL/N obtained after 30 to 40 passages of N-AL/ N in culture. It was negative for murine leukemia virus when tested by the complement-fixation (CF) test. (For CF [COMUL] tests, 20% cell extracts were tested against "broad reactive" rat serum obtained from rats bearing transplantable Moloney sarcoma 501 virus-induced tumors. The tests were kindly performed by H. C. Turner.) ST-AL/N-T is a cell line derived from a tumor induced in a syngeneic mouse with ST-AL/N cells. Py-AL,/N (clone 3) is a derivative of N-AL/N cells transformed in vitro by polyoma virus. SV-AL/N was derived from N-AL/N cells transformed in vitro by the small plaq ue-forming variant of SV40. The N-Balb-3T3 and Balb-3T12 cell lines and derivatives were obtained from S. Aaronson, M. Martin, H. Smith, and G. Todaro. N-Balb-3T3 is a nontumorigenic cell line established from mouse embryos of the Balb/c strain (3). ST-Balb-3T3-TA31 Cl 1 and 17 Cl 1 are "spontaneously" transformed derivatives of N-Balb-3T3. Balb-3T12 and Balb-3T12-4 were also derived from Balb mouse embryos. They grow to higher saturation densities than N-Balb-3T3 and both are tumorigenic. Balb-3T12-4 releases murine leukemia virus spontaneously (1). SV-Balb-3T3-1 1A-8, T2, and lOAC1 7-J4 were derived from N-Balb-3T3 transformed in vitro by SV40. MSV-Balb-3T3-24 and MSV-Balb-3T3-K were derived from N-Balb-3T3 transformed in vitro by MSV. MSV-Balb-3T3-24 releases MSV and murine leukemia virus. MSV-Balb-3T3-K shows no evidence of production of either virus. However, addition of murine leukemia virus results in rescue of the MSV genome (2). Balb mouse embryo cell cultures were prepared in this laboratory and used after one or two passages. MT-Balb is a cell line established in this laboratory from a tumor induced in a Balb mouse by murine mammary tumor virus. Py-Ts-a-3T3, obtained from M. Vogt, was derived from the Swiss-3T3 line transformed by the Ts-a mutant of polyoma virus described by M. Fried. Polyoma virions are produced by these cells at the permissive temperature (31 C). L2, a subline of NCTC Cl 929 of Earle's C3H mouse L-cell strain, was obtained from S. Dales. Vero cells were purchased from the American Type Culture Collection. The L929 (CCLl) cell line employed for interferon assay was obtained from S. Baron. All of the cell lines were tested frequently for mycoplasmas and reinitiated from frozen stocks when contamination was detected.
The growth medium for all cells was Dulbecco medium supplemented with unheated fetal bovine serum at a concentration of 10%7c. All media contained penicillin (100 to 250 units/ml) and streptomycin (100,.g/ml).
Viruses. The A59 strain of mouse hepatitis virus prepared in NCTC-1469 cells was supplied by J. Parker. It was triply plaque-purified before use. Murine leukemia virus (Moloney strain) was obtained from J. Hartley and W. Rowe. This stock preparation did not contain MSV. Mengo virus was supplied by L. Caligiuri; reovirus type 3 by P. Gomatos; and vesicular stomatitis virus (VSV), Sindbis, and Chikungunya viruses by C. Buckler and S. Baron. Stocks of these viruses were prepared in L2 and Vero cell types. Preparation and assay of GDVII virus were performed as described previously (18).
Plaque titrations. Plaque assays were performed by inoculating virus which was diluted in 10or 5-fold steps in Dulbecco medium with 10% fetal bovine serum onto cell monolayers in 60-mm tissue culture dishes, 0.5 ml per dish. Virus was allowed to adsorb for 1 hr (2 hr for reovirus) at 37 C in 5% Co2 in humidified air with gentle tilting every 10 to 20 min. Three plates were used for each dilution. After the adsorption period, the inoculum was removed, and each plate received 8 ml of an overlay medium consisting of 0.95%/O Noble agar in Eagle minimal essential medium with 5%-;o unheated fetal bovine serum. A59 virus plaques on L2 cells were counted after incubation for 2 days at 37 C. With other virus-cell systems, 4 ml of a second overlay containing 0.005 to 0.01% neutral red was added 12 to 24 hr before plaques were enumerated. A59 virus plaques were counted at 4 or 5 days in Py-AL/N; VSV, at 3 or 4 days in L2; Mengo virus and reovirus, at 7 to 10 days in L2; and Sindbis, at 7 to 10 days in Vero cell types. Plaques of A59 virus in other mouse cell systems were counted between 3 and 14 days after infection (see Results).

RESULTS
Host-dependent differences in plaque production by A59 virus. Plaque production by the A59 strain of mouse hepatitis virus has been investigated with related normal (untransformed), "spontaneously" transformed, and virus-transformed cell types of murine origin. The results obtained with AL/N cell lines indicate that plaque formation by A59 virus is facilitated in virus-transformed cells.
A59 virus produced minute, indistinct plaques in normal, nontumorigenic cells (N-AL/N) and in "spontaneously" transformed, tumorigenic cells (ST-AL/N and ST-AL/N-T). Virus plaques were not detected until 7 or more days after infection. These plaques were less than 4 mm in diameter at 12 to 14 days.
Plaque formation was facilitated on cells transformed by polyoma virus (Py-AL/N) or SV40 (SV-AL/N). A59 virus produced plaques at least 4 mm in diameter by 3 to 5 days, and the titers were 2to 10-fold higher in virus-transformed than in untransformed and "spontaneously" transformed AL/N cells.
A59 virus plaques on virus-transformed AL/N cells have a distinctive appearance. They are red and distinguished by a light halo (Fig. 1, left). These plaques are turbid in character. The central region of each plaque contains both dead cells and infected cells which accumulate increased amounts of neutral red. The diminished accumulation of neutral red at the periphery by cells most recently infected is transient (Fig. 1, right). sorption, multiplication, and release, from virusinduced cytopathogenicity, or from interferon production. These features of A59 virus infection have been investigated.
Thermal stability of A59 virus, adsorption, and A59 virus immunofluorescence. A59 virus derived from both N-AL/N and Py-AL/N cells had a half-life of approximately 1 hr at 37 C and 10 min at 45 C in the growth medium.
The rates of adsorption of A59 virus measured by plaque production on Py-AL/N cells were the same for virus produced in N-AL/N and Py-AL/N cells. By 1 hr at 37 C, the level of A59 virus adsorbed by Py-AL/N cells was about 60%c of that obtained at 3 hr. The rates of virus adsorption by plaque production on N-AL/N cells were not measured because the plaques were indistinct and could not be enumerated accurately.
As a measure of the rate and efficiency of the early events of infection, the accumulation of A59 virus-specific antigen was determined in N-AL N and Py-AL/N cells by indirect fluorescent-antibody staining. In both cell types, discrete foci of cytoplasmic fluorescence were observed 5 hr after infection. At  Monolayers of N-AL/N, Py-AL/N, and ST-AL/N-T cell types were inoculated with A59 virus at multiplicities of 70, 7, 0.7, and 0.07. After adsorption for 1 hr at 37 C, the monolayers were washed three times and reincubated at 37 C with Dulbecco medium supplemented with 10% fetal bovine serum. At 2-hr intervals, from 4 to 36 hr after infection, media were collected from cultures of each type which had been infected at each VOL. 6,1972 503 -AA '14 AL.
k, R STURMAN AND TAKEMOTO multiplicity; samples were frozen rapidly and stored at -90 C for assay. The first increase in A59 virus was detected in the interval between 6 and 8 hr after infection (Fig. 2). The kinetics of A59 virus production were similar in N-AL/N, Py-AL/N, and ST-AL/ N-T cell types. At multiplicities from 0.07 to 70, A59 virus production during the first 16 hr after infection was fiveto sixfold higher in Py-AL/N cells than in N-AL/N cells. During the same interval, virus production in ST-AL/N-T cells was 35to 45-fold lower than in N-AL/N cells.
Cell-associated A59 virus was also measured at frequent intervals. Differences of similar magnitude were found between titers of both intracellular and released virus from N-AL/N, Py-AL/N, and ST-AL/N cells.
The differences between A59 virus yields from cultures infected at lower multiplicities became larger at later times. By 36 hr in cultures infected at a multiplicity of 0.07, the yield of A59 virus from Py-AL/N cells was 40 times that from N-AL/N cells. The cumulative yield from ST-AL/N-T cultures infected at the same multiplicity was more than 120-fold lower than that from of Py-AL/N cells, compared with less than 1% of N-AL/N cells. Except for small numbers of syncytia, virus-induced cytopathological changes did not become apparent in all AL/N cell types until 12 to 24 hr after maximal levels of A59 virus production were reached.
Interferon production and interference induced by A59 virus in N-AL/N and Py-AL/N cells. Confluent monolayers were infected with A59 virus at a multiplicity of 5 PFU/cell. Uninfected cultures and cultures exposed to heat-inactivated Chikungunya virus served as controls. Media were collected at 24 hr, incubated for 5 days at 4 C at pH 3, neutralized, and assayed for interferon by the method of GDVII virus yield reduction in L 929 cells. The cell monolayers 24 hr after infection were challenged with VSV or Sindbis virus. Media were collected 18 hr later and assayed for these viruses.
Not With several viruses, cytopathological changes were more marked in one of the cell types. This was the case with Sindbis and VSV in N-AL/N and with reovirus in Py-AL/N cells.

A59 virus infection of normal and transformed
Balb cells. The specificity of facilitation of A59 virus plaque formation and virus multiplication were further tested with normal and transformed Balb-3T3 and 3T12 cell lines. A59 virus plaques were obtained on normal Balb-3T3 (N-Balb-3T3) cells only irregularly, even 10 or more days after infection, whereas, on both "spontaneously" transformed (ST-Balb-3T3-17Cl 1, TA31CI 1, Balb-3T12, and Balb-3T124) and virus-transformed (SV-Balb-3T3-1OA Cl 7-J4, MSV-Balb-3T3-24, and MSV-Balb-3T3-K) Balb cells, A59 virus plaques were evident by 2 to 3 days. Some SV40-transformed Balb-3T3 cell lines (SV-Balb-3T3-11A-8 and T2) could not be maintained adequately with an agar overlay. Under liquid medium, however, virus-induced cytopathogenicity and the rate of spread of infection determined by immunofluorescence were significantly greater in these cells than in N-Balb-3T3. In all three cell types, the first increase in A59 virus was detected between 6 and 8 hr after infection. A59 virus production was considerably higher in both SV40-transformed and "spontaneously" transformed Balb-3T3 cells. The extent of the difference was related to the multiplicity of infection. By 12 hr after infection at a multiplicity of 70, A59 virus production was threeto sixfold lower in N-Balb-3T3 cells. With infection at a multiplicity of 0.07, the differences in A59 production measured at 12 hr were 100fold or greater between the normal and transformed Balb-3T3 cell types.
These data indicate that the disparity between A59 virus multiplication in normal and virustransformed cells is independent of strain differencesbetweencells ofAL/N and Balb mouse genotypes.
DISCUSSION The normal and transformed cells tested were all susceptible to A59 virus infection. This is shown by the progressive nature of virus-induced cytopathological changes, the spread of virus antigen synthesis detected by immunofluorescence, and the confluence of plaque formation at high multi-VOL. 6,1972  plicities of infection. The growth of A59 virus, however, has been found to be host-dependent. Approximately fivefold greater virus yields per cell were obtained during the first cycle of infection of virus-transformed AL/N cell hosts.
In Balb-3T3 cells, larger differences, 100-fold or greater, were found during the first cycle of infection at low multiplicities. This is probably the result of the rapid spread of infection among transformed Balb cells through syncytia formation. The syncytia-inducing capacity of A59 virus in these cells facilitates the production of virus plaques.
As revealed by the growth curves and by immunofluorescence, the rate of spread of A59 virus infection differs also in the normal and virustransformed AL/N cells. A59 virus titers at later intervals from AL/N cells cannot be directly correlated with virus yields per cell, since the number of cells participating in the later cycle and the duration of A59 virus production by cells infected earlier are not known.
Marked differences have been noted between "spontaneously" transformed AL/N and Balb-3T3 cells as hosts of A59 virus. The ST-AL/N-T cell is a poorer host for A59 virus than even the N-AL/N cell type. In contrast, ST-Balb-3T3 cells resemble SV40-transformed Balb-3T3 cells in supporting high levels of A59 virus production and syncytia formation. The Balb-3T3 cell line has been shown to contain the genetic information for an oncornavirus (4). The presence of this genome may play a role in determining the phenotypic character of the "spontaneously" transformed Balb-3T3 derivatives.
A59 virus growth is presumed to be independent of the production of infectious transforming virus for the following reasons: (i) no infective SV40 or polyoma virus virions are produced by mouse cells transformed by these viruses, (ii) A59 virus plaque size and virus growth are not affected by the state of MSV production in MSV-transformed cells, and (iii) A59 virus plaques of similar large size are obtained at both permissive and nonpermissive temperatures in a cell line transformed by the TS-a mutant of polyoma virus (Py-Ts-a-3T3).
The role of the transforming viruses in the response of the transformed cell hosts is not known. Investigations are in progress on the chemistry and biosynthesis of coronaviruses which may provide the basis for understanding the mechanism responsible for these differences in A59 virus growth.