J. COW. PATH.

1979.Vo~.89.

MORPHOGENESIS CULTURED OPTIMAL

281

OF FER-DE-LANCE VIRUS AT SUB(23%) AND SUPRACELL GROWTH TEMPERATURES

(FDLV) (36°C)

P. D. LUNGER University

of Delaware,

JVewark, DE 19711, U.S.A.

and H F. CLARK The Wistar InMute, Philadelphia, PA 19104, C:.S.A

INTRODUCTION

FDLV, a Fer-de-Lance (Bothrops a&ox) derived myxovirus, has been recentl) characterized at the biological and compositional levels (Clark, unpublished observations; Waters, Koprowski, Clark and Lunger, 1976). In reptilian cell lines the virus has an optimal growth temperature of approximately 30 “C, a temperature well below that of mammalian cells, and it is restricted in grolqth at 37 “C. Structural events associated with FDLV maturation in 4 reptilian cell lines at optimal growth temperature were described previously (Lunger and Clark, 1979). of ectoWide cell growth temperature ranges, aflorded by the utilization thermic cell lines, provide a unique opportunity to study infectious processes. The present report is concerned with the morphogenesis ofFDLV at pronounced sub- and supraoptimal cell growth temperatures. MATERIALS

AND

METHODS

Reptilian cell cultures, virus source and EM techniques were described previousI) monolayers, used in the klunger and Clark, 1979). Mammalian cell (BHK/2lj present study, were grown in Eagle’s minimal essential medium supplemented with 10 per cent foetal calf serum. Maintenance medium for virus-infected growth medium containing 2 per cent foetal calf serum.

cells wa\

RESULTS

Events concerning morphogenesis associated with FDLV infection were studied at suboptimal (23 “C) and supraoptimal (36 “C) cell growth temperatures in the 4 reptilian lines utilized in the preceding report, and suboptimall) (36 “C) in one mammalian cell line (BHK/2 1). (23’- and 30 “C) and optimally At 23 “C Rsf and VH-3 cells appeared normal, but Rss and BHK/21 cells 0021-9975/79/020281+11$02.00/O

0

1979 Academic

Press Inc.

(London)

Limited

“82

P. 1). I~LrNGEl:

.4SD

II

1:.

CI.AiIli

were markedly rounded. At 36 CI VH-3, Rsf and KHKj21 \\(rc r~ormal ill appearance, but Rss showed cxtcnsivc cell rounding. Table 1 gives the relative quantities of budding virus, cytoplasmic~ IIII~~CY)caFsid and cytoplasmic inclusion bodies in reptilian and mammaliarr (.(.I1 lines at sub- and supraoptimal cell growth temperatures. Drtcrminations I\ WI’ made by thin-section EM examination of FDLV-infected ccl1 samples :rt I 1 days post-infection. Of the reptilian lines only rattlesnake fibroma cells produced large amounts of virus at suboptimal (23 ‘(2) growth tcmperaturc. x

Cell line

Temp.

Budding

oirus

Cq’toplasmic nucleocapsid

GE* RSf

23‘

RSS

VH-3 BHK/2 1 30-

BHK/2 1

36

GE Et:: VH-3 BHK/2 1

* For abbreviations

See text.

TAII1.E 2 COMPARATIVE SIZE OF PDLV CULTURED IN REPTILIAN AND MAMMALIAN OPTIMAL GROWTH TEMPERATURES. NUMBERS IN PARENTHESES REFER

Temp. 23’

30” 36”

Cell line

Spheroidal oirions nm Mean diam. Max. diam.

GE Rsf RSS VH-3 BHK/Z 1

208 (2)

BHK/2 1

193 (22)

GE Rsf RSS VH-3 BHK/P 1

211 (6) -

CELL LINES AT SUB- AND SIIPRATO NUMBER OF VIRIONS .MEAS1!HDD

I+‘ilamentous ciriom nm Mean len,yth L4fa.x. length

263 351 246 281

627 116)

965

830 17)

1088

280

437 (13)

877

281

1053 (2)

1579

REPTILIAN

MYXOVIRUS

MORPHOGENESIS

AT

23

AND

36 “c:

2%

did the mammalian BHK/21 line at 30 “C. Cytoplasmic nucleocapsid ~vas present in significant quantities only in gecko embryo cells at 23 “C. Table 2 shows the mean and maximum diameter of spheroidal and filamentous virions produced by the various cell lines at sub- and supraoptimal temperatures. Since the numbers of virions in some samples were low, determination of statistical significance between groups was unfeasible. Table 2 also shows the relative numbers of spheroidal and filamentous virions observed. Gecko Embryo (GE) at 23°C Infected gecko embryo cells at this temperature showed a high degree of cytoplasmic blebbing. Such extensions commonly contained membrane. systems, sometimes definable as Rough endoplasmic reticulum (Rer) cisternac, and occasionally mitochondria. On rare occasions nucleocapsid strands were present within the bleb cytoplasm (Fig. 1). Gecko embryo cells produced comparatively few budding viruses. The! generally appeared at the tip of a microvillus-like plasma membrane extension (Fig. 2), and morphologically showed features characteristic of myxoviruses. Occ:asionally spheroidal released virions were observed. No filamentous forms, tither attached or released, were seen. The cells contained large amounts of cytoplasmic nucleocapsid visualized as strands or tubules measuring 16 nm in diameter (Fig. 3). This dimension was characteristic of the other strandcontaining reptilian cell lines regardless of cell incubation temperature. (1ontrol cells showed a lesser degree of plasmalemmal blebbing than infected ones. Aside from this feature and the absence of virus-related products, there were no structural differences at the visual level between the two cell groups. Gecko Embryo (GE) at 36 ‘C Neither virions nor cytoplasmic nucleocapsid were found in infected gecko embryo cells. However, these cells contained relatively small amounts of specific cytoplasmic inclusion bodies. Control cells did not differ morphologically from infected ones. Rattlesnake Fibroma (Rsj-) at 23 “C Plasma membranes were characterized by frequent, narrow microvilluslike extensions. These often contained a spheroidal budding virion at theit distal end (Fig. 4). Nucleocapsid strands as visualized in transverse section were not usually well defined within budding virions. Recognizable cytoplasmic nucleocapsid strands were not visualized. Filamentous forms of FDLV were also prominent in these cells (Fig. 5). Mixed populations of spheroidal and filamentous particles were frequent in extracellular spaces (Fig. 6). (Control cells showed the same general cytological features as infected 011~. Rattlesnake Fibroma (Rsf) at 36 “C infected

Rsf cells at 36 “C showed

wide variation

in plasma

membrane

i7 000. Fig. 1. GE cells at 23 CL C:ytoplasmic blebs often wntain nucleocapsid strands. Fig. 2. GE cells at 23 Cl. Presumptive visions firqurntly appear at thv tip ofrni[,~o\-illlls-likr ( !IO~~;I~IIII( 79 800. extensions. Fig. 3. GE cells at 23 C. Cytoplasmic nucleocapsid strands measure 16 nm in diamc.tw. Str;~nd~ generally occupy large regions of the cytoplasm at this temperature. 57 000. Fig. 4. Rsf cells at 23 ‘C. \‘irions are seen budding distally from cytoplasmic cxtcnsions. .>7 000. Fig. 5. Rsf cells at 23 ‘C. A filamentous profile of FDLV is shown. ,I 57 000. Fig. 6. Rsf cells at 23 C. A mixture ofspheroidal and filamentous viral profiles is commonly ohservctl. x 57 000.

REPTILIAN

MYXOVIRUS

MORPHOGENESIS

AT

23

AND

36

“c:

285

Fig. 7. Rsf cells at 36 “C. Budding and released \iriollr arc scr’tl. (:ytoplasmic nuclrocapsid is also present. .< 57 000. Fiq. 8. Rsf cells at 36 “C. A rarely observed filamentous viral profilr is situated between two cytoplxsmil, extensions. i 57 000. Fig. 9. Rss cells at 23 “C. Spheroidal virions, are shown. .~ 79 800. Fig. 10. As Fig. 9 showing club-shaped virion. % 79 800. Fig. 11. VH-3 cells at 23 ‘C. Spheroidal, released virions arc located within a vacuole. 57 000. Fig. 14. VH-3 cells at 23 ‘C. Budding spheroidal and filamrntous profiles appear in approximatrl~ equal numbers. x 57 000.

286

I’.

I).

LUNGER

AND

11 F. CLXREi

extension morphology ranging from club-shaped to microvillus-like. < lu t)shaped forms frequently contained a single expanded Rer cistcrna \vith flocculent internal material. Virus production was not extensive in these cells. LYhen virion budding did occur, it was generally at the distal portion of narrow cytoplasmic cxtcnsions (Fig. 7). Much of the cytoplasm of the cell (in the lower portion of the micrograph) contained nucleocapsid strands. A rarely observed filamentous lorm is illustrated in Fig. 8. Control cells contained a pronounced expansion of Rer cisternae compared to infected ones, and in some cells mitochondria were greatly swollen. Rattlesnake Spleen (Rss) at 23°C’ Most plasma membrane extensions of infected Rss cells were microvillus-like, although occasional expanded forms were also noted. The cytoplasm was characterized by numerous specific inclusion bodies. Virus production was infrequent. Budding occurred from narrow plasma membrane extensions. Virion morphology was variable, ranging from spheroidal (Fig. 9) to bulbous or fiIamentous (Fig. 10). No cytopiasmic nuclrocapsid strands were observed. Control cells showed a moderate degree of plasmalemmal blebbing, but few microvillus-like formations. Occasional extracellular membranes were present in the vicinity of the plasma membrane. Specific cytoplasmic inclusions were absent in these cells. Rattlesnake Spleen (Rss) at 36°C These cells were characterized by large cytoplasmic processes extending from the plasma membrane. Each process usually contained a single, highlyexpanded Rer cisterna with moderately dense internal material. Myelin-like figures were closely adjacent to plasma membranes in the extracellular spaces of some cells. No evidence of virus production or viral-related products was observed. Control cells at this temperature did not differ structurally from infected ones. Viper Heart (KY-3)

at 23 “C

Infected VH-3 cells were characterized by many microvillus-like plasma membrane extensions, a moderate amount of Rer, many orthodox mitochondria, and numerous vacuoles containing varying amounts of heterogeneous internal material. These cells produced moderate-to-large amounts of FDLV at this temperature. Unlike at optimal cell growth temperature, aberrant synthesis was not observed. Most viral profiles appeared spheroidal (Fig. 11) although occasional filamentous forms were seen (Fig. 12). Cytoplasmic nucleocapsid strands and specific inclusion bodies were not observed. Control VH-3 cells did not differ structurally from infected ones.

REPTILIAN

MYXOVIRUS

MORPHOGENESIS

AT

2:s AND

36

‘c:

287

Fig. 13. BHK/21 cells at 30 “C. Spheroidal viral profiles show typical myxovirus morphology. :s 57 000 Fig. 14. As Fig. 13 showing filamentous viral profile. i( 57 000. 1:i.g. 15. VH-3 cells at 30 “C. Portions of three specific cvtoplasmic inclusion bodies show characteristic highly-ordered internal membrane structure. 75 800 ,< .

288

I’.

D. LUNGER

AND

H F. CLARK

Viper Heart (VH-3) at 36 “C Infected cells showed few cytoplasmic extensions and vacuoles compared to uninfected ones. They also displayed no evidence of virus production, including absence of nucleocapsid strands and inclusion bodies. Control cells, therefore: were not examined.

BHh* at 23 “C Infected cells showed plasma membrane extensions primarily of the thin or microvillus-like variety. Occasional oval myelin-like figures were associated with the plasma membrane surface. Some cells displayed large aggregations of cytoplasmic filaments arranged in semiparallel array. There was no visual Control cells did not differ structurally from evidence of virus production. infected ones.

BHI;‘ at 30 ‘C Infected cells showed a prominence of short, club-like plasma membrane extensions, some of which were involved in virus production. Occasional areas of cytoplasmic nucleocapsid strands were found in peripheral regions. Individual strands measured 15.80 nm in width, a dimension similar to that of reptilian cell-associated strands. Virus production was extensive, and profiles consisted of both spheroidal and filamentous forms in approximately equal numbers (Figs 13 and 14). Viral nucleocapsid was generally well defined as tubules in transverse action, or as elongated densities in tangential section. Control cells did not differ structurally from infected ones. Examination of FDLV-infected BHK/2I cells incubated at near-optimal (36 “C) cell growth temperature failed to reveal the presence of virions or virus-related products.

CytoplasmicInclusion Bodies Specific cytoplasmic inclusion bodies were membrane-bound organelles often closely associated with nucleocapsid strands (Fig. 15). They were not present in uninfected cells. They were found in varying amounts in all reptilian cell lines, depending on temperature, but not in BHK/21 cells. Inclusions had a mean diameter of 907 nm (5 1 inclusions measured). There was no correlation between inclusion size and cell growth temperature. Inclusions also did not differ structurally with respect to host-cell origin. Their internal composition consisted of highly-ordered membrane systems, sometimes resembling compact myelin figures, situated in a moderately dense background matrix. Other areas of the inclusion (regions not consisting of compact myelin) also contained lamellar systems arranged in semiparallel array. Membranes here were 7 nm in width, and were separated by a 17.50 nm space. In certain planes of section a narrow (4.40 nm) zone of intermediate density was found between membranes. Occasionally membranes appeared to be interconnected by perpendicular filaments regularly repeating at 8.80 nm intervals.

REPTILIAN

MYXOVIRUS

MORPHOGENESIS

AT

23

AND

36” c:

289

DISCUSSION

The preceding communication dealt with FDLV morphogenesis at near optimal cell growth temperature (30 “C) , whereas the present one is concerned with these events occurring at sub- and supraoptimal temperatures. Such wide variations in cell growth temperature (23 ’ to 36 “C) have not been commonly utilized in cell-virus interaction studies at the fine structure level. Hence, ectothermic cell lines provide a unique means of relating temperature diflerential to the infectious process. From the observations summarized in Table 1 it is apparent that both host cell origin and cell growth temperature are important considerations in regard to FDLV production. Host cell-mediated influence was indicated b\ low yields of GE- and Rss-associated virus in comparison to high yields if Rsf- and VH-3-associated virus at 23 “C. Furthermore, no virus was detected in FDLV-infected BHK/21 cells at that temperature. If the overall relative amount of budding virus is considered in the four the two rattlesnake cell lines reptilian cell lines, regardless of temperature, displayed greater capacity for virion production than the viper heart and gecko embryo cell lines. This observation is not surprising in view of the fact tha.t Fer-de-Lance is more closely related phylogenetically to rattlesnakes than to Asian pit vipers (VH-3) or to geckos (GE). Geckos are more distantly relitted to Fer-de-Lance than pit vipers, and cells derived from the former produced the least amount of virus of all the cell lines. FDLV-infected pit viper-derived cells showed an intermediate amount of budding virus (between gecko and rattlesnake). It is not possible to postulate a mechanism to explain virus replication in a host as distant as mammals (BHK/21), although the concept of specific viral tropism in vitro has long since been discarded. 0f all the lines examined at various temperatures only Rsf cells producrd virus at suboptimal, optimal and supraoptimal temperatures. This may reflect a cell-mediated effect, possibly related to a transformed cell state. As far as cell growth temperature is concerned, it is clear that FDLV replicates more efficiently at suboptimal than at supraoptimal temperatures in reptilian cells. BHK/21 cells at suboptimal temperature (30 C) also, produced considerable quantities of virus. At supraoptimal temperatures no, virus was produced in any cell line with the single exception of a rclativell few particles budding from Rsf cells. At suboptimal temperatures there appeared to be an inverse relationship between the presence of budding virus and the amount of cytoplasmic nucleocapsid strands. Infected gecko cells showed large amounts of nucleocapsid, but. little budding virus; Rsf and VH-3 showed large and moderate degrees of virion production, respectively, but not cytoplasmic nucleocapsid. At 30 -C: a small amount of nucleocapsid strands and large quantities of virus wcrr found in BHK/21 cells. At supraoptimal temperatures the production of cytoplasmic nucleocapsid strands was inhibited in all cell lines, with the exception of a small amount found in Rsf cells.

290

I’.

D.

LUNGER

AiKD

11 1:. (:LARK

The mean diameter of spheroidal virions \t’as 1~ at sul~ol~timal 1lia11 ,I! optimal temperatures in all cell lines cscept GE. Ho\ve\-cr, (hr samples size. itI the latter >+as so small (\2 particles measured) that the clifftircnc,tt ma) trot t)(, significant or valid. Likewise, the numbers of filamentous forms mcasur~d WW’ too low to attempt comparisons. When spheroidal suboptimal and optimai temperature-derived virions are pooled separately from all reptilian ccl1 1int.s and compared to each other statistically by the Studellt’s t-test, tllc‘ size, difference is significant at the P -< 0.01 level. The overall mean dian1ctc.r Of suboptimal virions is 183 nm (h’ :-= 63), and that of optimal \.irions is 230 11111 (N =.=44). Only minor ultrastructural differences existed between infected and controi cells. Control GE cells at 23 “C sho\ved a lesser degree of plasmalemmal blebbing than infected ones. This suggests that blebbing facilitates or enhances virion production as indicated in other mysovirus studies (Cornpans, Holmes, Dales and Choppin, 1964; Feller, Dougherty and DiStefano, 1969). At 36 ‘C! control Rsf cells display more extensive Rer cisternal expansion than infected ones. The functional significance, if any, of this observation is obscure. The functional significance of reptilian cell-associated cytoplasmic inclusion bodies is not yet understood. They were present only in in&ted cells, and hence are considered to be specific. Furthermore, they xvere usually located in thost regions of the cell containing cytoplasmic nucleocapsid strands. \\%ilr man) types of cytoplasmic inclusion bodies are associated Lvith mysoxrirus-infcctcd avian and mammalian cells (Armstrong, Pereira and Valentine, 1962; Cornpans and Dimmock, 1969; Compans, Harter and Choppin. 1967; Cornpans, Dimmock and Meier-Ewert, 1970; Kopp, Kempf and Kroeger, 1968; Norrby, Chiarini and Marusyk, 1970; Porebska, Pereira and Armstrong, 1968; Saito. Yoshioka, Igarashi and Nakagawa, 1970; 5lorrongiello and Dales, 1977; Shaw and Compans, 1978) none resemble structurally the highly-ordered membrane arrangement found in the reptilian mysovirus-ccl1 system. OLII. inclusions may represent lysosomes or phagosomes. Compact myelin-containing bodies have been described as such in a \?rus-producing murine carcinoma ccl1 line (Arnold, Soule and Russo, 1976). On the other hand, lysosome-like organelles are generally thought to be more structurally hetcrogcncous in regard to internal composition than our inclusions. Ultrastructural histochemical studies in progress should elucidate the functional nature of these. bodies. The results of these studies are generally in agreement hvith previous observetions that virus temperature requirements for replication are of predominant importance in determining the efficiency of virus replication in systems Lvhere the virus and host cell have different temperature optima (Clark and Karzon, 1968; Clark and Soriano, 1974). SUMMARY

FDLV, a recently isolated reptilian myxovirus, replicates at suboptimal cell growth temperature in one mammalian and four reptilian cell lines. At supraoptimal temperature only rattlesnake fibroma cells produced virus. Virion

REPTILIAN

MYXOVIRUS

MORPHOGENESIS

six, cytoplasmic nucleocapsid strands, bodies are considered on a comparative temperature differentials.

AT

23

AND

36 “c

291

and specific virus-related inclusion basis with respect to host cell and

REFERENCES

Armstrong, J. A., Pereira, H. G., and Valentine,

R. C. (1962). Morphology and development of respiratory syncytial virus in cell cultures. JVature, 196,1179-l 181. Arnold, W. J.. Soule, H. D., and Russo, J. (1976). Fine structure of a murinc mammary carcinoma cell line. In Vitro, 12, 57-64. Clark, H. F., and Karzon, D. T. (1968). Temperature optima of mammalian and amphibian viruses in cell cultures of homeothermic and poikilothermic origin. Archiv fiir die gesamte Virusforschung, 23, 270-279. Clark, H. F., and Soriano, E. Z. (1974). Fish rhabdovirus replication in non-piscine, ccl1 culture: new system for the study of rhabdovirus-cell interaction in which the virus and cell have different temperature optima. Infection and Immunity, 10, 180-188. Compans, R. LY., and Dimmock, N. J. (1969). An electron microscope study 01. single-cycle infection of chick embryo fibroblasts by influenza virus. Ti’rolqq, 39,499-515. Cornpans, R. W., Dimmock, N. J., and Meier-Ewcrt. H. (1970). An electron microscope study of the influenza virus-infected ~~11.In The Biology of Large R,li.-1 Viruses, pp. 87-108. Compans, R. W., Harter, D. H., and Choppin, P. W. (1967). Studies on pneumonia virus of mice (PVM) in cell culture. II. Structure and rnorphogenesis of Chc’ virus particle. Journal of Exkerimental >Wedicine, 126, 267-276. Cornpans, R. W., Holmes, K. V., Dales, S.. and Choppin, P. W. (1964). An ekC~r~Jr1 microscope study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5. l%ology, 30, 411 -426. Fcllcr, U., Dougherty, R. M., and DiStefano, H. S. (1969). Morphogenesis of NDV in chorioallantoic membrane. Journal qf Virology, 4, 753-762. Kopp, J. V., Kempf, .J. E., and KroeGcr, A. V. (1968). Cytoplasmic inclusions ol)scrvcd by electron microscopy m late influenza virus infection of chick embryo fibroblasts. Virology, 36, 681-683.

Lunger, P. D., and Clark, H F. (1979). Morphogenesis of Fcr-de-Lance virus (FDLV) cultured at optimal (30 “C) cell growth temperature. Journal of (,‘omparatiz Pathology, 89, 265-279. Morrongiello, M. P., and Dales, S. (1977). Characterization of cytoplasmic inclusions fijrmed during influenza/WSN virus infection of chick embryo fibroblast ccllx Iulcrvirobgy, 8, 281-293. Norrby, E., Chiarini, A., and Marusyk, H. (1970). Measles virus variants: intracellular appearance and biological characteristics of virus products. In T/~P Biology of’I,arge RNA Viruses, pp. 141-152. Par-cbxka, A., Pcreira, H. G., and Armstrong, J. A. (1968). Cytoplasmic inclusions and nuclear changes in cells infected with influenza-B viruses. Jollrtxtl ~~/‘~Ifedical ,llicrobiology, 1, 145-152. Saito, Y., Yoshioka, I., Igarashi, Y., and Nakagawa, S. (1970). Nuclear inclusions ohserved by electron microscopy in Cjnomolgus monkey kidney crllq infpctvd with influenza virus. Vi’irology, 40, 408-410. Sh‘tw, M. 11’., and Compans, R. W. (1978’). Isolation and characterization 01‘ cytoplasmic inclusions from influenza A virus-infccted cells. Journal qf Virolo,p: 25, 608-615. Waters, D. J., Koprowski, H., Clark, H. F., and Lunger. P. D. (1976). The ultrastructure, nucleic acid and polypeptide composition of Fer-de-Lance virus (FDLV). .‘lmerican S’ociet_yof Microbiolog, Abstracts, p. 224. [Receivedfor publication,

August 7th, 19781