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Viruses and renal carcinoma of Rana pipiens
VIROLOGY
41, 596-602
(1970)
Viruses
and
IX. The Influence
Renal
of Temperature
Frog Polyhedral MANETH Laboratory
of Virology
and
Carcinoma
GRAVELL
Deoxyribovirus
AND
St. Jude Accepted
Pipiens
and Host Cell on Replication
Cytoplasmic
Immunology,
of Rana
ALLAN
Children’s March
of
(PCDV)’
GRANOFF
Research
Hospital,
Memphis,
Tennessee
26, 1970
PCDV replication was studied over the temperature range 8”33” in cells originating from warmand cold-blooded vertebrates of t.hree classes: fish, mammals, and birds. These cells were fathead minnow (FHM), baby hamster kidney (BHK), and chicken embryo (CEF) .No significant virus replication occurred in any cell type at either 8” or lo”, but at 12” replication occurred in all cell types used. However, yields of PCDV and rate of production at 12” were greater in cells from cold-blooded than from warm-blooded vertebrates (FHM > BHK > CEF). Furthermore, the viral latent, period at 12” was shorter in FHM cells (12-24 hours) than in either BHK (2-3 days) or CEF cells (34 days). As the temperature was raised from 12 to 30 or 31°, the viral latent period decreased, the rate of multiplication increased, and high titers of infectious virus were obtained over a broad temperature range in all cells. At 30” to 31”, there was little difference in the latent periods (FHM cells, 3-4 hours; BHK cells, 4-6 hours) or in the rates of PCDV replication in cells originat)ing from poikilotherms or homeotherms. Irrespective of cell type, both yield and rate of PCDV replication were redrlced at 32”. Infectious progeny virus was not made in any cell type at 33”, but virus-specific macromolecules were formed at t,his temperature. Data from temperature shift experiments (33.5” -+ 24”) suggested that a t,emperature-sensitive event occurred late in the PCDV replication cycle, possibly at assembly of viral components. These results indicate that yields of PCDV and rate of replication are regulated by the host cell, but the permissive t)emperatnre zone is controlled by viral genes.
belong to a single major serotype (Lehane et al., 1968; Came et al., 196X; Iiaminski et az.~ 1g6g). Previous studies have shown that PCDV multiplies in a wide variety of cells derived from both poikilothermic and homeothermic vertebrates [fish, amphibia, reptiles, birds, and mammals (Granoff, 1969)]. Clark and Iiareon (1968) reported that PCDV did not replicate in turtle heart cells at 4” but replicated slowly at 14”. Further, they reported that turtle heart, primary chick embryo, and primary rhesus monkey kidney cells supported virus growth at 23” and 30”, but not at 36”. Granoff and co -workers (1966) found that virus adsorption and
INTRODUCTION
Isolates of a polyhedral cytoplasmic deoxyribovirus (PCDV) which differ physitally and chemically from poxviruses have been obtained from normal and Luck6 tumor-bearing leopard frogs (Rana pipiens) (Granoff et al., 1965, 1966; Clark et al., 1968)) from newts (Triturus viridescens) (Clark et al., 1969), and from bullfrog tadpoles (Rana catesbeiana) (Wolf et al., 1968). By immunological criteria these virus isolates, obtained in widely separated geographic areas of the United States, appear to 1 Supported by Public Health Grant 5 ROl CA 07055 from the Institute and by ALSAC.
Service National
Kesearch Cancer 596
EFFJ
OF
TEMPERATURE
penetration occurred in fish and chick cells at 33”, but infectious virus was not made at this temperature. Additionally, when they transferred infected cultures from 24” (permissive temperature) to 33”, irrespective of time after infection, and incubation was continued, virus titers did not increase significantly over those found at the time of transfer to 33”. They concluded from this finding that some temperature-sensitive event occurred late in the multiplication cycle. In this study we have made use of the broad phylogenetic susceptibility of cells to PCDV infection to determine the influence of temperature and host cell on the rate of production and yields of progeny virus. A preliminary report of this work has appeared elsewhere (Granoff and Gravell, 1968).
ON
PCDV
RF:PLICATION
597
Chemical Rubber Co., Cleveland, Ohio) using input multiplicities of l-10 PFU~cell. Virus inocula were adsorbed to cell monolayers for 1 hour at the respective incubation temperatures, except where indicated otherwise, and the residual input virus removed by washing cell cultures twice Jvith pH 7.2 phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). Temperatures of the PBS wash medium coincided with the incubation temperatures of the various virus growth curves. Eagle’s minimum essential medium (Grand Island Biological Co., Grand Island, Ken- York) in Hanks’ salt solution was used in vial cultures (1 ml/culture) and the same medium in Earle’s salt solution was used for petri dish cultures (5 ml/culture). Both media were supplemented with fetal calf serum (Hyland Lab., Los Angeles, California) to a final concentration of 2 % (-1IE1\1-2). Conditions MATERlALS AND METHODS of incubation are described in detail under Cell cultures. Cells originating from three Results as they pertain to particular expericlasses of vertebrates (fish, birds, and mamments. Petri dish cultures lvere incubated mals) were used. Two cell lines originated in a gaseous environment (5 o/o COa-95% from freshwater teleost fishes, the fathead air) with temperature controlled to f l”, and minnow (P%zephaZes pronzelas) (FHhI) vial cultures were immersed in electrically (Gravel1 and Malsberger, 1965) and the heated water baths with temperature conbluegill (Lepomis ~~~acrochi~~s)(BGL) (Gratrolled to =tO.25”. Infected petri dish culvell and Malsberger, unpublished). Cells tures were sampled by dislodging adhering of mammalian and avisn origin, respectively, cells with a rubber policeman, and harvestmere baby hamster kidney 21, clone 13 ing the combined cells and medium in (BHK) (Stoker and Rfacpherson, 1964), and disposable plast’ic tubes. Cell-associated primary chick embryo cells (CEF). Methods virus (CAV) was released into the medium of cell cultivation were as described preby treating for 1 minute at maximum power viously (Gravel1 and Malsberger, 1965; with a 10 MC Raytheon Sonic Oscillator. Granoff et al., 1966; Stoker and Macpherson, CAV was released from infected vial cul1964). tures by placing the sample vials directly 17irus ad preparation of virus stocks. into the sonicator cup and treating for 1 PCDV isolates used in this study were FV 1 minute. Thus, titers represent total virus and FV 3 (Granoff et al., 1965, 1966). When yield (CAV plus released virus). compared according to morphological, bioVirus assay. Yields of infecbious virus were logical, chemical, and immunological criteria determined by plaque assay as previously, (Granoff et al., 1966; Lehane et cd., 196s; described (Granoff et al., 1966; JIaes et al. Came et al., 196s; Kaminski et al., 1969), 1967) in monolayers of FHM cells grown in FV 1 and FV 3 are indistinguishable, and 60 X 15 mm petri dishes. therefore, will simply be referred to as I’CDV. RESULTS Virus growth curves. Virus growth curves Influence of Host Cell on the LoweT Permissive were obtained by infecting cells grown as Temperature Limit, Rate of Replicutior~, monolayers in either 60-mm plastic petri and Yields of Progeny I’irus dishes (Falcon Plastics, Los Angeles, California) or the interior bottom surface of To determine whether cell origin had an 1’3.mm glass vials (“Titeseal” vials, The influence on either the low temperature
i59s
FIG. arrows
GItAVELL
1. The indicate
AND
effect of incubation at 8” on replication the transfer of cultures to 25”.
limit permissive for PCDV multiplication, or on the rate and quantities of infectious virus produced, cell cultures derived from tissues of homeothermic and poikilothermic vertebrates were inoculated with PCDV (vial cultures, 1-3 PFU/cell) and incubated at various temperatures. At intervals after infection, samples were withdrawn, sonicated, and assayed for yields of infectious virus. As shown in E’ig. IA and B, no significant increase in infectious virus was detected at 8” in cells originating from either a poikilotherm (FHM) or a homeotherm (BHK) over incubation periods of 14 and 24 days, respectively. Failure of either cell type to produce infectious virus could not be attributed to loss of cell viability resulting from the low incubation temperature. When FH_11 or BHK cultures were transferred at various times after infection from S” to 22, a temperature permissive for PCDV replication, 30.fold or greater increases in infectious virus were detected (Fig. IA and B). Likewise, FH,\I and BHK cells incubated at 10” over a 20-day period did not produce significant quantities of infectious PCDV. In contrast to the results of the S” and 10” experiments, at 12’ replication of PCDV occurred in PHJI, BHK, and CEF cells. Furthermore, the length of the virus latent period and the rate of infectious virus production were dependent upon the host cell, the latent period being shortest and the rate of progeny virus formation greatest in cells derived from a poikilotherm (FHM, Fig. 2A). The virus latent period in FHJI cells incubated at 12” n-as between l:! and 24 hours
GIIANOFF
of PCI)V
in FHM
FIG. 2. Growth in FHM,
BHK,
(A)
and
BHK
(B)
of PCDV at 12” (A) and CEF cells.
cells.
The
and 24”
(B)
with approximately lo7 PFU/ml detectable 3 days after infection. The virus latent period and kinetics of PCDV replication in another cell line derived from a poikilotherm (BGL) were essentially the same as found in FHM cells (data not shown). In cells of homeothermic origin, BHK and CEF, the virus latent periods at 12’ were 2-3 and
EFFECT
OF
TEMPERATURE
ON
3-4 days, respectively. A titer of 10’ l’FU,‘ml of PCDV was not detected in BHK cells incubated at 12” until between 7 and 10 days after infection, whereas only lOG PPU/‘ml was detected on the tenth day after infection of CEF cells. The variations in rate of replication and yields of PCDV from FHM, BHK and CEE’ cultures incubated at 12O cannot be attributed to differences in cell number. All cultures, regardless of type, contained similar numbers of cells, and as will be shown subsequently, produced essentially the same titer of infectious virus when incubated at higher permissive temperatures. In E’HRI, BHK, and CEF cultures incubated at 24” the same relative order in rate of PCDV multiplication was found as at 12”, i.e., FH?rI > BHK > CEF (Fig. ‘2B). Nevertheless, noteworthy differences exist between virus growth curves obtained at 12’ and 24”. First, the 24” viral latent periods were reduced and the rates of infectious virus production were increased, when compared to the 12” growth curves, for all three cell types. Note that now the viral latent periods ranged only from 6 to 10 hours, whereas at 12” they ranged from 12 hours to 4 days. l;urthermore, maximal virus yields from FHM, BHK, and CEF cultures did not differ significantly and were reached by 4s hours after infection. Thus, at 24”, although infectious virus n-as detected earlier in cells derived from a poikilotherm (FHM), the final yields of virus from cultures of homeothermic or poikilothermic origin were esscnt,ially the same.
FIG.~~.
Growth
of PCDV
in FHM
(A)
and
BHK
PCDV
599
REPLICATION
Injtuence of Host Cell on the I:pper I’emissive Temperature Limit Figures 3A and B show single-step PCDV growth curves (vial cultures, 3 PFU/cell) obtained in FHM and BHK cell cultures incubated within the temperature range 24” to 33”. As the incubation temperature was raised over the range 24” to 30”, the viral latent periods decreased in both FHhI and BHK cells. This point is illustrated more strikingly in BHK cells (Fig. 3B) than in FHM cells (Fig. 3A). Even though the viral latent periods of the 25” and 27” growth curves in FHJI cells mere similar (4-5 hours), higher titers of PCDV were detected at earlier times postinfection in cultures incubated at 27”. Thus, more frequent sampling would probably have show the viral latent period to be shorter at 27”. In cells derived from poikilotherms and homeotherms, the viral latent period was shortest in cultures incubated at about 30” (FHM, 3-4 hours; BHK, 4-G hours). Final yields of infectious virus were similar within a broad temperature zone, irrespective of cell type. PCDV replicated to maximum titer in FHllI cells from about 18” to 31”, and in BHK and CEl: cells from at least 24”-31”. In FHhI or BHK cells, no difference was evident betiveen the yields and rate of PCDV replication at 30” or 31”, but the rate of production decreased and yields of infectious virus wre reduced br\ TOM0 % in both cell types at 32” (data not, shown). No measurable increase in infectious virus over background levels was det.ected in FHM and BHK cultures incubated at 33”.
(B)
cells
at temperatures
between
25”
and
33”.
600
GRAVELL
AND
GRANOFF
Results in agreement with the above, although not shown, were also obtained with infected CEF cells. Therefore, host cell does not appear to determine the upper permissive temperature for PCDV replication. Stepdown PCDV Growth Curves from Nonpermissive (SS.S”) to Permissive Temperature (24”) The purpose of the following experiment was to determine whether events leading to infectious virus production can occur at 33.5”, a temperature nonpermissive for PCDV replication. Infection was synchronized by adsorbing PCDV (2 PFU/cell) to FHM monolayers in 19-mm vials for 1 hour at 4”. Virus adsorption, but not penetration, occurs at 4’ (Granoff et al., 1966). Infected cultures were then transferred to 33.5” where virus adsorption and penetration, but not infectious virus production, occur (Granoff et al., 1966). After 30 minutes for virus penetration, the unadsorbed inoculum was removed and the cultures were washed twice with PBS prewarmed to 33.5”. At intervals thereafter, groups of infected cultures were transferred from 33.5 to 24” and incubation continued at this temperature. Following transfer to 24”, samples were taken at various intervals over the next 12 hours. Virus yields were also determined at various times from cultures held continually at 33.5” for a la-hour period. If events leading to infectious virus production occur at 33.5”, then the viral latent period should decrease in relation to the time infected cultures are incubated at 33.5” before transfer to 24”. Thus, relative to the 24” growth curve, infectious virus should be detected at progressively earlier times until the temperature-sensitive event in the multiplication cycle is reached. Incubation at 33.5” for periods up to 3 hours prior to transfer to 24” resulted in progressively higher titers of infectious virus starting at 3 hours post-24” incubation and continuing for an additional 5-8 hours (Fig. 4). Incubation of infected cultures at 33.5” for time periods in excess of 3 hours (4-6 hours) yielded growth curves closely coinciding with the growth curve obtained for 3 hours’ preincubation at 33.5” (data not shown).
FIG. 4. The effect of preincubation at 33.5” (nonpermissive temperature) on subsequent multiplication of PCDV at 24” (permissive temperature). Infected FHM cultures were held at 33.5” for the times indicated above, transferred t,o 24”, and incubation continued at this temperature for up to 12 hours. Growth curves began upon transfer of cultures to 24”, wit’h the exception that control cultures were held at 33.5” for 12 hours to show that progeny virus was not made under these conditions.
Thus, incubation of infected FHM cells for 3 hours at 33.5’ prior to transfer to 24” reduces the virus latent period to between 2 and 3 hours, a reduction of about 2 hours when compared to the viral latent period in FHM cells incubated only at 24” (Fig. 2B). Although infect.ious progeny were not made at 33.5’, viral DNA, RNA, and protein were formed based upon the finding of Feulgen (viral DNA) and immunofluorescent (viral protein) positive cytoplasmic inclusions (Granoff, 1969)) and RNA hybridizable with viral DNA (Gravel& unpublished data). Therefore, these data, coupled with those of experiment,s previous temperature-shift (Granoff et al., 1966), suggest that a temperature-sensitive event occurs late in the PCDV replication cycle, possibly at assembly of viral components. DISCUSSION
An extensive literature is available regarding the effects of temperature on replication of viruses of warm-blooded vertebrates (Lwoff, 1969; Carmichael and Barnes, 1969). In general, viruses of homeotherms replicate optimally within a narrow temperature zone
which correlates closely with the normal body temperature of their host. At temperutures above or below this optimum zone, ultimate yields of infectious virus fall off markedly. Conversely, it appears that viruses of poikilotherms replicate with maximal yield over a broader range of t,emperatures, which compose a higher proportion of the permissive temperature zone, than do viruses of homeotherms. Furthermore, viruses of poikilotherms which have been studied replicate within most of their permissive temperature zones at a rate proportional to their incubation temperature; the rate of replication being greatest slightly below the temperature where virus replication ceases. In support of these conclusions, we have found that ?-ields of PCDV from FHI\I cells are maximal over the temperature range IS”-31”. At 12”, the lowest temperature at which PCDV replicated, final yields of PCDV were reduced only about SS % of maximum. In the upper permissive temperature zone, maximal yields and rate of replication of PCDV were obtained at 30”-31”, yields were reduced 70-80 % at 32”, and no detectable virus multiplication occurred at 33”. Other viruses of poikilotherms also behave similarly, since yields of infectious pancreatic necrosis (IPN) (Wolf et al., 1960) and Egtved viruses (Zwillenberg and Zwillenberg, 1964), pathogens of trouts with reovirus-like (Moss and Gravell, 1969) and rhabdovirus-like (Zwillenberg et al., 1965) characteristics, respectively, were reduced only 3- to 4-fold of maximum when grown in RTG-2 cells (Wolf and Quimby, 1962) incubated at 4” (Kenneth Wolf, personal communication). With IPN virus, no significant difference in yields was detected in FHM cells incubated from 10” to 25”, and the rate of virus replication paralleled incubation temperature (Gravell, unpublished). In these studies the upper permissive temperature limit for IPN virus replicat,ion was not determined, but no virus replication was detected in FHRI cells incubated at 33”. Finally, yields of sockeye salmon virus were maximal between S” and 20”, with an approximate 90 % reduction in yield at 4”. Replication of sockeye salmon virus did not occur at 23” (Wingfield et al., 1969).
One might 110~ ask why it is that viruses of poikilotherms replicate with maximum yield over a wider range of temperatures than do viruses of homeotherms. A plausible explanation is that enzymes coded for by viruses of poikilotherms, for survival value, must function at a variety of temperatures, since body temperatures of their hosts fluctuate according to ambient conditions. The slower replication of PCDV at low permissive temperatures in cells of homrotherms (BHK, CEF) than in those of poikilotherms (FHM, BGL) can be similar13 explained. Virus reproduction depends upon use of the metabolic and protein-synthesizing machinery of the infected cell. Body temperatures of homeot’herms are maintained within rather narrow limits. Therefore, it is not unreasonable to assume that rate of PCDV replication at low temperature would be greater in cells of poikilotherms, since they are adapted to function over a range of temperatures. Although temperature influenced the rate of PCDV replication in cells originating from warm- and cold-blooded vertebrates, the temperature zone permissive for PCDV replication 1~:~sindependent of cell origin. Xeither cells derived from poikilotherms or homeotherms produced infectious virus at incubation temperatures of 10” or 33”, whereas, regardless of cell origin, infectious progeny were produced betlveen 12” and 32”. These results raise the question whether the inability of PCDV to replicate at 10” or 33” is virus or cell controlled. As previously mentioned, IPN virus replicated to maximal titer in FHlI cells at lo”, while PCDV failed to replicate under these conditions. Thus, these data suggest that the inability of PCDV to replicate in lgHI\I cells at 10” is virus, rather than cell, controlled. Furthermore, the cell lines derived from poikilotherms used in this study (FHM, BGL) have temperature optima for growth near that of cells of homeotherms. Both PHhI and BGL cells replicate maximally at approximately 35” (Gravel1 and Malsberger, 196.5; Gravell, unpublished data). Therefore, PCDV ceased to replicate at a temperature below that optimal for growth of all cells used in this study, suggesting that the upper
602
GRAVELI,
AND
temperature limit permissive for PCDV replication is also controlled by viral genes. The knowledge that a broad phylogenetic spectrum of susceptible cells is available which are more temperature tolerant than is the infecting virus should prove useful in the design of future experiments to further elucidate the steps involved in PCDV replication. A ACKNOWLEDGMENT Mr. Melvin assistance.
C. Smith
provided
skilled
technical
REFERENCES
CAME, P. E., GEERING, G., OLD, L. J., and BOYCE, E. A. (1968). A serological study of polyhedral cytoplasmic viruses isolated from amphibia. Virology 36, 392-400. CARMICHAEL, L. E., and BARNES, F. D. (1969). Effect of temperature on growth of canine herpesvirus in canine kidney cell and macrophage cultures. J. Infect. Dis. 120,664-668. CLARK, H. F., and KSRZON, D. T. (1968) Temperature optima of mammalian and amphibian viruses in cell cultures of homeothermic and poikilothermic origin. Arch. Gesamte Virusforsch. 23, 27S279. CLARK, H. F., BRENNAN, J. C., ZEIGEL, R. F., and KARZON, D. T. (1968). Isolation and characterization of viruses from the kidneys of Rana pipiens with renal adenocarcinoma before and after passage in the red eft (Triturus viridescens) . J. Viral. 2, 629440. CLARK, H. F., GRAY, C., FABIAN, F., ZEIGEL, R. F., and KARZON, D. T. (1969). Comparative studies of amphibian cytoplasmic virus strains isolated from the leopard frog, bullfrog, and newt. In “Recent Results in Cancer Research.” Springer, Berlin. DULBECCO, R., and VOGT, M. (1954). Plaque for. mation and isolation of pure lines with poliomyelitis. J. Ezp. Med. 99, 167-182. GRANOFF, A. (1969). Viruses of amphibia. Curr. Top. Microbial. Immunol. 50, 107-137. GRANOFF, A., and GRAVELL, M. (1968). Influence of temperatrue and host cell on replication of frog virus 3 (FV3) Bacterial. Proc. p. 178 (abstract). GRANOFF, A., CAME, P. E., and RAFFERTY, K. A., JR. (1965). The isolation and properties of viruses from Rana pipiens: Their possible relationship to the renal adenocarcinoma of the leopard frog. Ann. N. Y. Acad. Sci. 126,237-255. GRANOFF, A., CAME, P. E., and BREEZE, D. C.
GRANOFF (1966). Viruses and renal carcinoma of Rana pipiens. I. The isolat,ion and properties of virus from normal and tumor tissue. T’irolog?/ 29, 133-148. GRAVIELL, M., and MALSBERGXR, It. G. (1965). A permanent cell line from the fat,head minnow (Pimephales promelas). Ann. S. Y. Acad. Sci. 126, 555-565. KAMIXSKI,S., CLARK, H.F., and KARZOX,~). T. (1969). Comparative immune response to amphibian cytoplasmic viruses assayed by the complement-fixation and gel immunodiffusion tests. J. Immunol. 103, 260-267. LEHANE:, D. E., JR., CLARK, H. F., and KARZON, D. T. (1968). Antigenic relationships among frog viruses demonstrated by the plaque reduct,ion and neutralizat,ion kinetics tests. Virology 34, 590-595. LIVOFF, A. (1969). Death and transfiguration of a problem. Racteriol. Rev. 33, 390-403. MAES, R., GRBNOFF, A., and SMITH, W. R. (1967). Viruses and renal carcinoma of Rana pipiens. III. The relat,ionship between input multiplicity of infection and inclusion body formation in frog virus 3-infect,ed cells. Virology 33, 137-144. Moss, L. H., and GRAVF,LL, M. (1969). Ult,rastrurt,ure and sequentia1 development of infectious pancreatic necrosis virus. J. Viral. 3, 52-58. STOKER, M., and MACPRERXON, I. (1964). Syrian hamster fibroblast cell line BHK-21 and its derivatives. Nature 203, 1355-1357. WINGFIELD, W. H., FRYER, J. L., and PILCHER, K. S. (1969). Properties of the sockeye salmon virus (Oregonstrain). Proc. Sot. Exp. Biol. Med. 130, 1055-1059. WOLF, K., and QUIMBY, M. C. (1962). Established eurythermic line of fish cells in vitro. Science 135, 1065-1066. WOLF, K., SNIESZKO, S. F., DUNBAR, C. E., and PYLE, E. (1960). Virus nature of infectious pancreatic necrosis in trout. Proc. Ser. Erp. Biol. Med. 104, 105-108. WOLF, K., BULLOCK, G. L., DUNBAR, C. E., and QUIMFSY, M. C. (1968). Tadpole edema virus: A viscerot,ropic pathogen from anuran amphibians. J. Infect. Dis. 118,253-272. Z~ILLEN~FXG, L. O., and ZWILLENBERG, H. H.L. (1964). Elektronenmikroskopische Untersuchungen an Regenbogenforellen mit, infektioser Nierenschwellung und Leberdegeneration. Arch. Gesamte Virusjorsch. 14, 319-331. ZX~ILLENBERG, L. O., JENSEN, M. H., alld ZWILLENBERG, H. H. L. (1965). Electron microscopy of the virus of viral haemorrhagic septicaemia of rainbow trout (egtved virus). ilrch. Gesamte Virusforsch. Ii, l-19.
41, 596-602
(1970)
Viruses
and
IX. The Influence
Renal
of Temperature
Frog Polyhedral MANETH Laboratory
of Virology
and
Carcinoma
GRAVELL
Deoxyribovirus
AND
St. Jude Accepted
Pipiens
and Host Cell on Replication
Cytoplasmic
Immunology,
of Rana
ALLAN
Children’s March
of
(PCDV)’
GRANOFF
Research
Hospital,
Memphis,
Tennessee
26, 1970
PCDV replication was studied over the temperature range 8”33” in cells originating from warmand cold-blooded vertebrates of t.hree classes: fish, mammals, and birds. These cells were fathead minnow (FHM), baby hamster kidney (BHK), and chicken embryo (CEF) .No significant virus replication occurred in any cell type at either 8” or lo”, but at 12” replication occurred in all cell types used. However, yields of PCDV and rate of production at 12” were greater in cells from cold-blooded than from warm-blooded vertebrates (FHM > BHK > CEF). Furthermore, the viral latent, period at 12” was shorter in FHM cells (12-24 hours) than in either BHK (2-3 days) or CEF cells (34 days). As the temperature was raised from 12 to 30 or 31°, the viral latent period decreased, the rate of multiplication increased, and high titers of infectious virus were obtained over a broad temperature range in all cells. At 30” to 31”, there was little difference in the latent periods (FHM cells, 3-4 hours; BHK cells, 4-6 hours) or in the rates of PCDV replication in cells originat)ing from poikilotherms or homeotherms. Irrespective of cell type, both yield and rate of PCDV replication were redrlced at 32”. Infectious progeny virus was not made in any cell type at 33”, but virus-specific macromolecules were formed at t,his temperature. Data from temperature shift experiments (33.5” -+ 24”) suggested that a t,emperature-sensitive event occurred late in the PCDV replication cycle, possibly at assembly of viral components. These results indicate that yields of PCDV and rate of replication are regulated by the host cell, but the permissive t)emperatnre zone is controlled by viral genes.
belong to a single major serotype (Lehane et al., 1968; Came et al., 196X; Iiaminski et az.~ 1g6g). Previous studies have shown that PCDV multiplies in a wide variety of cells derived from both poikilothermic and homeothermic vertebrates [fish, amphibia, reptiles, birds, and mammals (Granoff, 1969)]. Clark and Iiareon (1968) reported that PCDV did not replicate in turtle heart cells at 4” but replicated slowly at 14”. Further, they reported that turtle heart, primary chick embryo, and primary rhesus monkey kidney cells supported virus growth at 23” and 30”, but not at 36”. Granoff and co -workers (1966) found that virus adsorption and
INTRODUCTION
Isolates of a polyhedral cytoplasmic deoxyribovirus (PCDV) which differ physitally and chemically from poxviruses have been obtained from normal and Luck6 tumor-bearing leopard frogs (Rana pipiens) (Granoff et al., 1965, 1966; Clark et al., 1968)) from newts (Triturus viridescens) (Clark et al., 1969), and from bullfrog tadpoles (Rana catesbeiana) (Wolf et al., 1968). By immunological criteria these virus isolates, obtained in widely separated geographic areas of the United States, appear to 1 Supported by Public Health Grant 5 ROl CA 07055 from the Institute and by ALSAC.
Service National
Kesearch Cancer 596
EFFJ
OF
TEMPERATURE
penetration occurred in fish and chick cells at 33”, but infectious virus was not made at this temperature. Additionally, when they transferred infected cultures from 24” (permissive temperature) to 33”, irrespective of time after infection, and incubation was continued, virus titers did not increase significantly over those found at the time of transfer to 33”. They concluded from this finding that some temperature-sensitive event occurred late in the multiplication cycle. In this study we have made use of the broad phylogenetic susceptibility of cells to PCDV infection to determine the influence of temperature and host cell on the rate of production and yields of progeny virus. A preliminary report of this work has appeared elsewhere (Granoff and Gravell, 1968).
ON
PCDV
RF:PLICATION
597
Chemical Rubber Co., Cleveland, Ohio) using input multiplicities of l-10 PFU~cell. Virus inocula were adsorbed to cell monolayers for 1 hour at the respective incubation temperatures, except where indicated otherwise, and the residual input virus removed by washing cell cultures twice Jvith pH 7.2 phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). Temperatures of the PBS wash medium coincided with the incubation temperatures of the various virus growth curves. Eagle’s minimum essential medium (Grand Island Biological Co., Grand Island, Ken- York) in Hanks’ salt solution was used in vial cultures (1 ml/culture) and the same medium in Earle’s salt solution was used for petri dish cultures (5 ml/culture). Both media were supplemented with fetal calf serum (Hyland Lab., Los Angeles, California) to a final concentration of 2 % (-1IE1\1-2). Conditions MATERlALS AND METHODS of incubation are described in detail under Cell cultures. Cells originating from three Results as they pertain to particular expericlasses of vertebrates (fish, birds, and mamments. Petri dish cultures lvere incubated mals) were used. Two cell lines originated in a gaseous environment (5 o/o COa-95% from freshwater teleost fishes, the fathead air) with temperature controlled to f l”, and minnow (P%zephaZes pronzelas) (FHhI) vial cultures were immersed in electrically (Gravel1 and Malsberger, 1965) and the heated water baths with temperature conbluegill (Lepomis ~~~acrochi~~s)(BGL) (Gratrolled to =tO.25”. Infected petri dish culvell and Malsberger, unpublished). Cells tures were sampled by dislodging adhering of mammalian and avisn origin, respectively, cells with a rubber policeman, and harvestmere baby hamster kidney 21, clone 13 ing the combined cells and medium in (BHK) (Stoker and Rfacpherson, 1964), and disposable plast’ic tubes. Cell-associated primary chick embryo cells (CEF). Methods virus (CAV) was released into the medium of cell cultivation were as described preby treating for 1 minute at maximum power viously (Gravel1 and Malsberger, 1965; with a 10 MC Raytheon Sonic Oscillator. Granoff et al., 1966; Stoker and Macpherson, CAV was released from infected vial cul1964). tures by placing the sample vials directly 17irus ad preparation of virus stocks. into the sonicator cup and treating for 1 PCDV isolates used in this study were FV 1 minute. Thus, titers represent total virus and FV 3 (Granoff et al., 1965, 1966). When yield (CAV plus released virus). compared according to morphological, bioVirus assay. Yields of infecbious virus were logical, chemical, and immunological criteria determined by plaque assay as previously, (Granoff et al., 1966; Lehane et cd., 196s; described (Granoff et al., 1966; JIaes et al. Came et al., 196s; Kaminski et al., 1969), 1967) in monolayers of FHM cells grown in FV 1 and FV 3 are indistinguishable, and 60 X 15 mm petri dishes. therefore, will simply be referred to as I’CDV. RESULTS Virus growth curves. Virus growth curves Influence of Host Cell on the LoweT Permissive were obtained by infecting cells grown as Temperature Limit, Rate of Replicutior~, monolayers in either 60-mm plastic petri and Yields of Progeny I’irus dishes (Falcon Plastics, Los Angeles, California) or the interior bottom surface of To determine whether cell origin had an 1’3.mm glass vials (“Titeseal” vials, The influence on either the low temperature
i59s
FIG. arrows
GItAVELL
1. The indicate
AND
effect of incubation at 8” on replication the transfer of cultures to 25”.
limit permissive for PCDV multiplication, or on the rate and quantities of infectious virus produced, cell cultures derived from tissues of homeothermic and poikilothermic vertebrates were inoculated with PCDV (vial cultures, 1-3 PFU/cell) and incubated at various temperatures. At intervals after infection, samples were withdrawn, sonicated, and assayed for yields of infectious virus. As shown in E’ig. IA and B, no significant increase in infectious virus was detected at 8” in cells originating from either a poikilotherm (FHM) or a homeotherm (BHK) over incubation periods of 14 and 24 days, respectively. Failure of either cell type to produce infectious virus could not be attributed to loss of cell viability resulting from the low incubation temperature. When FH_11 or BHK cultures were transferred at various times after infection from S” to 22, a temperature permissive for PCDV replication, 30.fold or greater increases in infectious virus were detected (Fig. IA and B). Likewise, FH,\I and BHK cells incubated at 10” over a 20-day period did not produce significant quantities of infectious PCDV. In contrast to the results of the S” and 10” experiments, at 12’ replication of PCDV occurred in PHJI, BHK, and CEF cells. Furthermore, the length of the virus latent period and the rate of infectious virus production were dependent upon the host cell, the latent period being shortest and the rate of progeny virus formation greatest in cells derived from a poikilotherm (FHM, Fig. 2A). The virus latent period in FHJI cells incubated at 12” n-as between l:! and 24 hours
GIIANOFF
of PCI)V
in FHM
FIG. 2. Growth in FHM,
BHK,
(A)
and
BHK
(B)
of PCDV at 12” (A) and CEF cells.
cells.
The
and 24”
(B)
with approximately lo7 PFU/ml detectable 3 days after infection. The virus latent period and kinetics of PCDV replication in another cell line derived from a poikilotherm (BGL) were essentially the same as found in FHM cells (data not shown). In cells of homeothermic origin, BHK and CEF, the virus latent periods at 12’ were 2-3 and
EFFECT
OF
TEMPERATURE
ON
3-4 days, respectively. A titer of 10’ l’FU,‘ml of PCDV was not detected in BHK cells incubated at 12” until between 7 and 10 days after infection, whereas only lOG PPU/‘ml was detected on the tenth day after infection of CEF cells. The variations in rate of replication and yields of PCDV from FHM, BHK and CEE’ cultures incubated at 12O cannot be attributed to differences in cell number. All cultures, regardless of type, contained similar numbers of cells, and as will be shown subsequently, produced essentially the same titer of infectious virus when incubated at higher permissive temperatures. In E’HRI, BHK, and CEF cultures incubated at 24” the same relative order in rate of PCDV multiplication was found as at 12”, i.e., FH?rI > BHK > CEF (Fig. ‘2B). Nevertheless, noteworthy differences exist between virus growth curves obtained at 12’ and 24”. First, the 24” viral latent periods were reduced and the rates of infectious virus production were increased, when compared to the 12” growth curves, for all three cell types. Note that now the viral latent periods ranged only from 6 to 10 hours, whereas at 12” they ranged from 12 hours to 4 days. l;urthermore, maximal virus yields from FHM, BHK, and CEF cultures did not differ significantly and were reached by 4s hours after infection. Thus, at 24”, although infectious virus n-as detected earlier in cells derived from a poikilotherm (FHM), the final yields of virus from cultures of homeothermic or poikilothermic origin were esscnt,ially the same.
FIG.~~.
Growth
of PCDV
in FHM
(A)
and
BHK
PCDV
599
REPLICATION
Injtuence of Host Cell on the I:pper I’emissive Temperature Limit Figures 3A and B show single-step PCDV growth curves (vial cultures, 3 PFU/cell) obtained in FHM and BHK cell cultures incubated within the temperature range 24” to 33”. As the incubation temperature was raised over the range 24” to 30”, the viral latent periods decreased in both FHhI and BHK cells. This point is illustrated more strikingly in BHK cells (Fig. 3B) than in FHM cells (Fig. 3A). Even though the viral latent periods of the 25” and 27” growth curves in FHJI cells mere similar (4-5 hours), higher titers of PCDV were detected at earlier times postinfection in cultures incubated at 27”. Thus, more frequent sampling would probably have show the viral latent period to be shorter at 27”. In cells derived from poikilotherms and homeotherms, the viral latent period was shortest in cultures incubated at about 30” (FHM, 3-4 hours; BHK, 4-G hours). Final yields of infectious virus were similar within a broad temperature zone, irrespective of cell type. PCDV replicated to maximum titer in FHllI cells from about 18” to 31”, and in BHK and CEl: cells from at least 24”-31”. In FHhI or BHK cells, no difference was evident betiveen the yields and rate of PCDV replication at 30” or 31”, but the rate of production decreased and yields of infectious virus wre reduced br\ TOM0 % in both cell types at 32” (data not, shown). No measurable increase in infectious virus over background levels was det.ected in FHM and BHK cultures incubated at 33”.
(B)
cells
at temperatures
between
25”
and
33”.
600
GRAVELL
AND
GRANOFF
Results in agreement with the above, although not shown, were also obtained with infected CEF cells. Therefore, host cell does not appear to determine the upper permissive temperature for PCDV replication. Stepdown PCDV Growth Curves from Nonpermissive (SS.S”) to Permissive Temperature (24”) The purpose of the following experiment was to determine whether events leading to infectious virus production can occur at 33.5”, a temperature nonpermissive for PCDV replication. Infection was synchronized by adsorbing PCDV (2 PFU/cell) to FHM monolayers in 19-mm vials for 1 hour at 4”. Virus adsorption, but not penetration, occurs at 4’ (Granoff et al., 1966). Infected cultures were then transferred to 33.5” where virus adsorption and penetration, but not infectious virus production, occur (Granoff et al., 1966). After 30 minutes for virus penetration, the unadsorbed inoculum was removed and the cultures were washed twice with PBS prewarmed to 33.5”. At intervals thereafter, groups of infected cultures were transferred from 33.5 to 24” and incubation continued at this temperature. Following transfer to 24”, samples were taken at various intervals over the next 12 hours. Virus yields were also determined at various times from cultures held continually at 33.5” for a la-hour period. If events leading to infectious virus production occur at 33.5”, then the viral latent period should decrease in relation to the time infected cultures are incubated at 33.5” before transfer to 24”. Thus, relative to the 24” growth curve, infectious virus should be detected at progressively earlier times until the temperature-sensitive event in the multiplication cycle is reached. Incubation at 33.5” for periods up to 3 hours prior to transfer to 24” resulted in progressively higher titers of infectious virus starting at 3 hours post-24” incubation and continuing for an additional 5-8 hours (Fig. 4). Incubation of infected cultures at 33.5” for time periods in excess of 3 hours (4-6 hours) yielded growth curves closely coinciding with the growth curve obtained for 3 hours’ preincubation at 33.5” (data not shown).
FIG. 4. The effect of preincubation at 33.5” (nonpermissive temperature) on subsequent multiplication of PCDV at 24” (permissive temperature). Infected FHM cultures were held at 33.5” for the times indicated above, transferred t,o 24”, and incubation continued at this temperature for up to 12 hours. Growth curves began upon transfer of cultures to 24”, wit’h the exception that control cultures were held at 33.5” for 12 hours to show that progeny virus was not made under these conditions.
Thus, incubation of infected FHM cells for 3 hours at 33.5’ prior to transfer to 24” reduces the virus latent period to between 2 and 3 hours, a reduction of about 2 hours when compared to the viral latent period in FHM cells incubated only at 24” (Fig. 2B). Although infect.ious progeny were not made at 33.5’, viral DNA, RNA, and protein were formed based upon the finding of Feulgen (viral DNA) and immunofluorescent (viral protein) positive cytoplasmic inclusions (Granoff, 1969)) and RNA hybridizable with viral DNA (Gravel& unpublished data). Therefore, these data, coupled with those of experiment,s previous temperature-shift (Granoff et al., 1966), suggest that a temperature-sensitive event occurs late in the PCDV replication cycle, possibly at assembly of viral components. DISCUSSION
An extensive literature is available regarding the effects of temperature on replication of viruses of warm-blooded vertebrates (Lwoff, 1969; Carmichael and Barnes, 1969). In general, viruses of homeotherms replicate optimally within a narrow temperature zone
which correlates closely with the normal body temperature of their host. At temperutures above or below this optimum zone, ultimate yields of infectious virus fall off markedly. Conversely, it appears that viruses of poikilotherms replicate with maximal yield over a broader range of t,emperatures, which compose a higher proportion of the permissive temperature zone, than do viruses of homeotherms. Furthermore, viruses of poikilotherms which have been studied replicate within most of their permissive temperature zones at a rate proportional to their incubation temperature; the rate of replication being greatest slightly below the temperature where virus replication ceases. In support of these conclusions, we have found that ?-ields of PCDV from FHI\I cells are maximal over the temperature range IS”-31”. At 12”, the lowest temperature at which PCDV replicated, final yields of PCDV were reduced only about SS % of maximum. In the upper permissive temperature zone, maximal yields and rate of replication of PCDV were obtained at 30”-31”, yields were reduced 70-80 % at 32”, and no detectable virus multiplication occurred at 33”. Other viruses of poikilotherms also behave similarly, since yields of infectious pancreatic necrosis (IPN) (Wolf et al., 1960) and Egtved viruses (Zwillenberg and Zwillenberg, 1964), pathogens of trouts with reovirus-like (Moss and Gravell, 1969) and rhabdovirus-like (Zwillenberg et al., 1965) characteristics, respectively, were reduced only 3- to 4-fold of maximum when grown in RTG-2 cells (Wolf and Quimby, 1962) incubated at 4” (Kenneth Wolf, personal communication). With IPN virus, no significant difference in yields was detected in FHM cells incubated from 10” to 25”, and the rate of virus replication paralleled incubation temperature (Gravell, unpublished). In these studies the upper permissive temperature limit for IPN virus replicat,ion was not determined, but no virus replication was detected in FHRI cells incubated at 33”. Finally, yields of sockeye salmon virus were maximal between S” and 20”, with an approximate 90 % reduction in yield at 4”. Replication of sockeye salmon virus did not occur at 23” (Wingfield et al., 1969).
One might 110~ ask why it is that viruses of poikilotherms replicate with maximum yield over a wider range of temperatures than do viruses of homeotherms. A plausible explanation is that enzymes coded for by viruses of poikilotherms, for survival value, must function at a variety of temperatures, since body temperatures of their hosts fluctuate according to ambient conditions. The slower replication of PCDV at low permissive temperatures in cells of homrotherms (BHK, CEF) than in those of poikilotherms (FHM, BGL) can be similar13 explained. Virus reproduction depends upon use of the metabolic and protein-synthesizing machinery of the infected cell. Body temperatures of homeot’herms are maintained within rather narrow limits. Therefore, it is not unreasonable to assume that rate of PCDV replication at low temperature would be greater in cells of poikilotherms, since they are adapted to function over a range of temperatures. Although temperature influenced the rate of PCDV replication in cells originating from warm- and cold-blooded vertebrates, the temperature zone permissive for PCDV replication 1~:~sindependent of cell origin. Xeither cells derived from poikilotherms or homeotherms produced infectious virus at incubation temperatures of 10” or 33”, whereas, regardless of cell origin, infectious progeny were produced betlveen 12” and 32”. These results raise the question whether the inability of PCDV to replicate at 10” or 33” is virus or cell controlled. As previously mentioned, IPN virus replicated to maximal titer in FHlI cells at lo”, while PCDV failed to replicate under these conditions. Thus, these data suggest that the inability of PCDV to replicate in lgHI\I cells at 10” is virus, rather than cell, controlled. Furthermore, the cell lines derived from poikilotherms used in this study (FHM, BGL) have temperature optima for growth near that of cells of homeotherms. Both PHhI and BGL cells replicate maximally at approximately 35” (Gravel1 and Malsberger, 196.5; Gravell, unpublished data). Therefore, PCDV ceased to replicate at a temperature below that optimal for growth of all cells used in this study, suggesting that the upper
602
GRAVELI,
AND
temperature limit permissive for PCDV replication is also controlled by viral genes. The knowledge that a broad phylogenetic spectrum of susceptible cells is available which are more temperature tolerant than is the infecting virus should prove useful in the design of future experiments to further elucidate the steps involved in PCDV replication. A ACKNOWLEDGMENT Mr. Melvin assistance.
C. Smith
provided
skilled
technical
REFERENCES
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GRANOFF (1966). Viruses and renal carcinoma of Rana pipiens. I. The isolat,ion and properties of virus from normal and tumor tissue. T’irolog?/ 29, 133-148. GRAVIELL, M., and MALSBERGXR, It. G. (1965). A permanent cell line from the fat,head minnow (Pimephales promelas). Ann. S. Y. Acad. Sci. 126, 555-565. KAMIXSKI,S., CLARK, H.F., and KARZOX,~). T. (1969). Comparative immune response to amphibian cytoplasmic viruses assayed by the complement-fixation and gel immunodiffusion tests. J. Immunol. 103, 260-267. LEHANE:, D. E., JR., CLARK, H. F., and KARZON, D. T. (1968). Antigenic relationships among frog viruses demonstrated by the plaque reduct,ion and neutralizat,ion kinetics tests. Virology 34, 590-595. LIVOFF, A. (1969). Death and transfiguration of a problem. Racteriol. Rev. 33, 390-403. MAES, R., GRBNOFF, A., and SMITH, W. R. (1967). Viruses and renal carcinoma of Rana pipiens. III. The relat,ionship between input multiplicity of infection and inclusion body formation in frog virus 3-infect,ed cells. Virology 33, 137-144. Moss, L. H., and GRAVF,LL, M. (1969). Ult,rastrurt,ure and sequentia1 development of infectious pancreatic necrosis virus. J. Viral. 3, 52-58. STOKER, M., and MACPRERXON, I. (1964). Syrian hamster fibroblast cell line BHK-21 and its derivatives. Nature 203, 1355-1357. WINGFIELD, W. H., FRYER, J. L., and PILCHER, K. S. (1969). Properties of the sockeye salmon virus (Oregonstrain). Proc. Sot. Exp. Biol. Med. 130, 1055-1059. WOLF, K., and QUIMBY, M. C. (1962). Established eurythermic line of fish cells in vitro. Science 135, 1065-1066. WOLF, K., SNIESZKO, S. F., DUNBAR, C. E., and PYLE, E. (1960). Virus nature of infectious pancreatic necrosis in trout. Proc. Ser. Erp. Biol. Med. 104, 105-108. WOLF, K., BULLOCK, G. L., DUNBAR, C. E., and QUIMFSY, M. C. (1968). Tadpole edema virus: A viscerot,ropic pathogen from anuran amphibians. J. Infect. Dis. 118,253-272. Z~ILLEN~FXG, L. O., and ZWILLENBERG, H. H.L. (1964). Elektronenmikroskopische Untersuchungen an Regenbogenforellen mit, infektioser Nierenschwellung und Leberdegeneration. Arch. Gesamte Virusjorsch. 14, 319-331. ZX~ILLENBERG, L. O., JENSEN, M. H., alld ZWILLENBERG, H. H. L. (1965). Electron microscopy of the virus of viral haemorrhagic septicaemia of rainbow trout (egtved virus). ilrch. Gesamte Virusforsch. Ii, l-19.