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Crystalline aggregates of infectious laryngotracheitis virus
VIHOLGGY
35, (1968)
Short Crystalline
Aggregates
Laryngotracheitis
Communications
of Infectious Virus
Since the first report by Bunting (1) of the occurrence of close-packed arrays of virus particles in cells of human skin papilloma, a number of animal viruses have been found to form crystals in viva. Among such viruses are poliovirus (2), coxsackicvirus A (S), coxsackievirus B (/t), echoviruses (5), reovirus (6), Shope papilloma virus (7), polyoma virus (8), SV40 virus (9), encephalomyocarditis virus (IO), adenoviruses (II, 12), and herpes simplex virus (13). It is t’he purpose of t’his communication to describe intranuclear crystalline formations of infectious lar\-ngot,racheitis (LT) virus observed in tissue culture cells. The source and maintenance of the strain of I,T virus used in the study have been described (14). Prior to t’his experiment, the virus was passed six times in chicken embryo kidney (CEK) tissue culture cells. Undiluted culture medium from the last passage harvested 72 hours after inoculation and having an infectivit,y titer of 1O3.6TCIDjo per 0.1 ml was used as inoculum in 0%ml quantitics. :\lonolayer cultures of CEK cells were grown in test tubes and 500-ml flat bottles in medium 199 (Difco) containing 100 units of penicillin and 100 pg of streptomycin per milliliter. The identity of the virus and the specificity of its cytopathic effect were confirmed by serum neutralization tests in tissue cultures :w previously reported (Id). Cell cultures were harvested 24, 36, 4S, and 72 hours after infection and fixed at 0” for an hour in a 1 ?Z solution of osmium tetroxide buffered at a pH 7.2. The preparations were then dehydrated in ascending grades of ethanol and embedded in Epon Sl2. Sections were cut with glass knives on an LT
cording to the method of Millonig (15), and examined with a Hitachi 11A electron microscope. Uninfected control culture cells, 5 days old, were processed according to the same method. At 72 hours after infection, approximately 30% of the cells contained varying numbers of LT virus particles and had pathologic changes ranging from mild degeneration to necrosis. Virus particles present in t,he nuclei were of nearly uniform size, measuring 870970 A, and had a single delimiting membrane, 100 A thick, but differed significantly in the structure of t’heir cores (lcig. 1). In about 40 % of all particles observed, the core was represented by a well defined, single body, 470-590 A in diameter, which was eit,her uniformly dense or had a central region of diminished opacity. Approximately 15 5%of the particles were devoid of the core. The remaining 45% of the virus particles contained, instead of a single core, 2 t’o 5 dense, irregularly shaped “granules” measuring 240-400 A. The “granules” tended to accumulate at the periphery of the particle, but one of them usually occupied a central position. The relative number of each form of virus part,icles varied considerably not, only among the nuclei but, also in different areas of the same nucleus; in some nuclei one form predominated (Figs. 1 and 2). All three forms of the particles are considered t)o be LT virus on the basis of the positive identification of the virus by serum neutralization tests and the observation that no such particles were present, in uninfected cont,rol culture cells Studies are nolv in progress to elucidate the funct’ional significance and mode of assembly of the particles. In approximately 2 % of the affected cells, virus particles had a tendency to aggregate in distinct linear arrays (Fig. I), or to form ordered aggregates possessing recognizable lattice patterns (Fig. 2). Such well ordered structures were observed only in t*he nuclei. 321
FIG:. 1. Litlear arrays of LT vinls particles iti the 1111c1er1s of tismle cultIn-e cell. Variatiotls are prwelll ill the sOrllct,we of the wre of virrm particles. X 58,500. FIG. 2. Close-park4 crystalline aggregate of LT virus part,icles having packilrg :wrangemellt srlggestive of a body-ceIit,ercd cubic lattice. X 57,500. 322
SHORT
323
COMMUNICATIONS
Serial sections revealed that the linear arrays (I:ig. 1) were space oriented, but lacked an identifiable crystal lattice structure. The aggregate of virus particles shown in Fig. 2 represents a plane through a crystalline formation. Although the parbicles appear to be closed-packed, their relationship indicates that each particle is surrounded by four nearest neighbors, suggesting that in a three-dimensional lattice t’he particles have eight, nearest neighbors. Such packing arrangement is consistent with a body-centered cubic lattice. This int,erpretation appears to be valid despite a slight angular dist)ortion of the crystal plane lvhich was estimated to be less than 6 degrees. The distortion n-as apparently caused by impact of t’he microtome knife during sect’ioning. As a result,, virus particles are slightly misshapen, but many of them st’ill have a clearly recognizable hexagonal profile. ACKNOWLEDGMENTS This work was supported in part, by research grant AI-3820 from the National Institut,e of Allergy and Infectious Ijiseases, United St,ates I’ltblir llralth Service, and the Universit,y of Illinois Agricult,ural Experiment Station, Regional Research Project NC-63. The assistance of hIrs. Marilyn Wingard in the preparation of t issne cultures is gratefully ackrrowledged. REFERENCES 1. BTNTISG, H., I’roc. Sot. E’~ptl. Riol. Med. 84, 327-332 (19.53). 2. STUART, 11. C., and FOGH, J., Exptl. Cell lies. 18, 378-381 (1!159). 3. N.\TTI:RK, C. F. T., IYirology 17, 520-532 (1962). 4. Xlo~c,.\s, C., Howr:, C., and ROSE, II. XLI., I’irology 9, 147-149 (1959). 5. ~tIFl
10. HINZ R. W., B.\RsM, G., and BMNH:~RD, W., E’xptl. Cell lies. 26, 371686 (1962). 11. KJE;LL~~N, L., LIGEKM.\LM, G., SvEDMYIi, It., K. G., Xature 15, 505-506 and THORSSON, (195.5) 1.8. J\IoRG.z~-, c., IIowr:, c., I~OSE, 11. >I., and ;\~OORI:, 1). Ii., .I. Biophys. ~?iochem. Cytol. 2, 351-360 (19.56). 13. MoRG.~, C., .JoNI~:s, E. P., I~oLD~:N, JI., and ROSE, IT. M., Virology 3, 368-371 (1938). 14. W,\TR.KH, A. hl., alld IIUSON, L. E., Proc. Sot. Erptl. Biol. Med. 112, 230-232 (1963). 15. MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736-739 (1961).
Department of Pathology and Hygiene College of Veterinary Medicine University of Illinois Urbana, Illinois Accepted March 25, 196X
Production Fixing
of a Potent Complement-
Murine
Leukemia
Serum from the Rabbit Reactions
with Various
Virus-Antiand its Types
of Tissue Culture Cells Complement-fixing (Cl;) sera have been obtained against avian leukosis viruses (ALV) in two different ways : from mammals carrying Rous sarcoma-virus (RSV)-induced tumors (I), and from rabbits immunized with degraded virus particles (2). High-titer sera have been obtained with the second method. Both types of sera proved to be direct’ed mainly against a group-specific inner component of t’he virus (3, 4). They have been used to develop ilz vitro infectivity tests as well as methods for diagnost,ic purposes (5, 6). JIurine leukemia viruses (NLV) behave similarly to ALV in various respect’s. CIC sera reacting lvit’h a group-specific 1ILV component, which in this case also appears t,o be an inrler structure of the virus particle (7), have been obtained recently from rats carrying tumors induced by *\\zI,V (8, 9). As in t,he case of ALV (2), ~vc have tried to produce such serum in rabbits by injection of degraded AIL\‘. The production procedure and the examination of the serum for viral
35, (1968)
Short Crystalline
Aggregates
Laryngotracheitis
Communications
of Infectious Virus
Since the first report by Bunting (1) of the occurrence of close-packed arrays of virus particles in cells of human skin papilloma, a number of animal viruses have been found to form crystals in viva. Among such viruses are poliovirus (2), coxsackicvirus A (S), coxsackievirus B (/t), echoviruses (5), reovirus (6), Shope papilloma virus (7), polyoma virus (8), SV40 virus (9), encephalomyocarditis virus (IO), adenoviruses (II, 12), and herpes simplex virus (13). It is t’he purpose of t’his communication to describe intranuclear crystalline formations of infectious lar\-ngot,racheitis (LT) virus observed in tissue culture cells. The source and maintenance of the strain of I,T virus used in the study have been described (14). Prior to t’his experiment, the virus was passed six times in chicken embryo kidney (CEK) tissue culture cells. Undiluted culture medium from the last passage harvested 72 hours after inoculation and having an infectivit,y titer of 1O3.6TCIDjo per 0.1 ml was used as inoculum in 0%ml quantitics. :\lonolayer cultures of CEK cells were grown in test tubes and 500-ml flat bottles in medium 199 (Difco) containing 100 units of penicillin and 100 pg of streptomycin per milliliter. The identity of the virus and the specificity of its cytopathic effect were confirmed by serum neutralization tests in tissue cultures :w previously reported (Id). Cell cultures were harvested 24, 36, 4S, and 72 hours after infection and fixed at 0” for an hour in a 1 ?Z solution of osmium tetroxide buffered at a pH 7.2. The preparations were then dehydrated in ascending grades of ethanol and embedded in Epon Sl2. Sections were cut with glass knives on an LT
cording to the method of Millonig (15), and examined with a Hitachi 11A electron microscope. Uninfected control culture cells, 5 days old, were processed according to the same method. At 72 hours after infection, approximately 30% of the cells contained varying numbers of LT virus particles and had pathologic changes ranging from mild degeneration to necrosis. Virus particles present in t,he nuclei were of nearly uniform size, measuring 870970 A, and had a single delimiting membrane, 100 A thick, but differed significantly in the structure of t’heir cores (lcig. 1). In about 40 % of all particles observed, the core was represented by a well defined, single body, 470-590 A in diameter, which was eit,her uniformly dense or had a central region of diminished opacity. Approximately 15 5%of the particles were devoid of the core. The remaining 45% of the virus particles contained, instead of a single core, 2 t’o 5 dense, irregularly shaped “granules” measuring 240-400 A. The “granules” tended to accumulate at the periphery of the particle, but one of them usually occupied a central position. The relative number of each form of virus part,icles varied considerably not, only among the nuclei but, also in different areas of the same nucleus; in some nuclei one form predominated (Figs. 1 and 2). All three forms of the particles are considered t)o be LT virus on the basis of the positive identification of the virus by serum neutralization tests and the observation that no such particles were present, in uninfected cont,rol culture cells Studies are nolv in progress to elucidate the funct’ional significance and mode of assembly of the particles. In approximately 2 % of the affected cells, virus particles had a tendency to aggregate in distinct linear arrays (Fig. I), or to form ordered aggregates possessing recognizable lattice patterns (Fig. 2). Such well ordered structures were observed only in t*he nuclei. 321
FIG:. 1. Litlear arrays of LT vinls particles iti the 1111c1er1s of tismle cultIn-e cell. Variatiotls are prwelll ill the sOrllct,we of the wre of virrm particles. X 58,500. FIG. 2. Close-park4 crystalline aggregate of LT virus part,icles having packilrg :wrangemellt srlggestive of a body-ceIit,ercd cubic lattice. X 57,500. 322
SHORT
323
COMMUNICATIONS
Serial sections revealed that the linear arrays (I:ig. 1) were space oriented, but lacked an identifiable crystal lattice structure. The aggregate of virus particles shown in Fig. 2 represents a plane through a crystalline formation. Although the parbicles appear to be closed-packed, their relationship indicates that each particle is surrounded by four nearest neighbors, suggesting that in a three-dimensional lattice t’he particles have eight, nearest neighbors. Such packing arrangement is consistent with a body-centered cubic lattice. This int,erpretation appears to be valid despite a slight angular dist)ortion of the crystal plane lvhich was estimated to be less than 6 degrees. The distortion n-as apparently caused by impact of t’he microtome knife during sect’ioning. As a result,, virus particles are slightly misshapen, but many of them st’ill have a clearly recognizable hexagonal profile. ACKNOWLEDGMENTS This work was supported in part, by research grant AI-3820 from the National Institut,e of Allergy and Infectious Ijiseases, United St,ates I’ltblir llralth Service, and the Universit,y of Illinois Agricult,ural Experiment Station, Regional Research Project NC-63. The assistance of hIrs. Marilyn Wingard in the preparation of t issne cultures is gratefully ackrrowledged. REFERENCES 1. BTNTISG, H., I’roc. Sot. E’~ptl. Riol. Med. 84, 327-332 (19.53). 2. STUART, 11. C., and FOGH, J., Exptl. Cell lies. 18, 378-381 (1!159). 3. N.\TTI:RK, C. F. T., IYirology 17, 520-532 (1962). 4. Xlo~c,.\s, C., Howr:, C., and ROSE, II. XLI., I’irology 9, 147-149 (1959). 5. ~tIFl
10. HINZ R. W., B.\RsM, G., and BMNH:~RD, W., E’xptl. Cell lies. 26, 371686 (1962). 11. KJE;LL~~N, L., LIGEKM.\LM, G., SvEDMYIi, It., K. G., Xature 15, 505-506 and THORSSON, (195.5) 1.8. J\IoRG.z~-, c., IIowr:, c., I~OSE, 11. >I., and ;\~OORI:, 1). Ii., .I. Biophys. ~?iochem. Cytol. 2, 351-360 (19.56). 13. MoRG.~, C., .JoNI~:s, E. P., I~oLD~:N, JI., and ROSE, IT. M., Virology 3, 368-371 (1938). 14. W,\TR.KH, A. hl., alld IIUSON, L. E., Proc. Sot. Erptl. Biol. Med. 112, 230-232 (1963). 15. MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736-739 (1961).
Department of Pathology and Hygiene College of Veterinary Medicine University of Illinois Urbana, Illinois Accepted March 25, 196X
Production Fixing
of a Potent Complement-
Murine
Leukemia
Serum from the Rabbit Reactions
with Various
Virus-Antiand its Types
of Tissue Culture Cells Complement-fixing (Cl;) sera have been obtained against avian leukosis viruses (ALV) in two different ways : from mammals carrying Rous sarcoma-virus (RSV)-induced tumors (I), and from rabbits immunized with degraded virus particles (2). High-titer sera have been obtained with the second method. Both types of sera proved to be direct’ed mainly against a group-specific inner component of t’he virus (3, 4). They have been used to develop ilz vitro infectivity tests as well as methods for diagnost,ic purposes (5, 6). JIurine leukemia viruses (NLV) behave similarly to ALV in various respect’s. CIC sera reacting lvit’h a group-specific 1ILV component, which in this case also appears t,o be an inrler structure of the virus particle (7), have been obtained recently from rats carrying tumors induced by *\\zI,V (8, 9). As in t,he case of ALV (2), ~vc have tried to produce such serum in rabbits by injection of degraded AIL\‘. The production procedure and the examination of the serum for viral