Exp. Path., Bd., 14 S. 9-15 (1977) Veterans Administration Hospital, trniversity of Arkansas for Medical Sciences, Little Rock, Arkansas and St. Louis University School of Medicine, St. Louis, Missouri U.S.A.
Human cell cultures infected by tumor-inducing and non-tumor-inducing viruses, an electron microscopic study By C. N.
SUN,
D. Hsu and H.
PINKERTON
With 6 figures (Received October 20, 1976) Key words: tumor-inducing viruses; non-tumor-inducing viruses; adenovirus; cell cultures: viral infection; virions; nuclear inclusions; human fibroblasts; viral specific antigens; electron microscopy
Summary This study is dealing with the infection patterns of adenovirus type 2 and type 12 on human fibroblast cell lines, KB, WI and MAF. With the exception of Ad-12 -+ WI, many intranuclear viral particles were present. None of these second passages (Ad-2 WI- WI, Ad-12 WI- WI, Ad-2 MAF-MAF, Ad-12 MAF-MAF) was found to have viral production. This indicates that the injections on both WI and MAF cells caused by both Ad-2 and Ad-12 cannot be serially transmitted. However, when these infected cells were used to expose KB cells, significant viral yields were obtained. This shows that the infected cells might still carry the viral specific antigens although no visible virions were observed. In regard to the hamster fibroblasts, the Ad-2 virus induces inclusions which show many intranuclear viral particles (as do other susceptible cells); the Ad-12 virus, on the other hand, only induces the early stages of nuclear inclusions, and no viral particles can be found (PINKERTON et al. 1966). Since it is obvious that type 12 adenovirus viral particles cannot be produced in the hamster cells, it is interesting to seek the differences between oncogenic (type 12) and nononcogenic (type 2) adenoviruses when they infect the human cells. The present research is dealing with the infection patterns of these two types of adenoviruses on two human fibroblast cell lines, WI and MAF. Comparative studies were made on the production of visible virions, the appearance of the mature nuclear inclusions, and all the abnormalities of the infected nuclei.
Material and methods Cell cultures and viruses Monolayers of three lines of human fibroblasts, KB, WI (originated from human-embryonic lung, Microbiological Associates, Bethesda, Maryland), MAF (derived from human skin and muscle fibroblasts) were cultured on plates with 1 x 106 cells per plate, cultures were transferred 3 or 4 days before infection was made, and were grown at 37°C in 5 ml of 2X Eagles' 10 % serum. All cell lines were infected separately with adenoviruses type 2 and type 12 (Ad-2 and Ad-12) at identical input multiplicities of 1-2 plaque forming units (PFU) per cell. Absorption of virus was carried out for 2 hours. After virus inoculum was removed, monolayers were washed twice overlayed with 2 % horse serum and incubated for 2 to 5 days, depending on the degree of cytopathology noted in the living cell monolayers. From each preparation, 2 plates were harvested and prepared for electron microscopy. Companion monolayers cultured on coverslips were infected in the same way for histo-cytological study. Coverslips were taken out of plates at the same intervals as were the plates pelleted for electron. microscopy, washed and removed the serum, fixed in absolute ethyl alcohol and stained with hematoxylin and eosin.
9
Second passages Second passages were made as follows: (a) Ad-2 ..... WI ..... WI. WI cells were infected with adenovirus type 2, which was propagated in WI line of cells. (b) Ad-12 ..... WI ..... WI. WI cells were infected with adenovirus type 12, which was propagated in WI line of cells. (c) Ad-2 ..... MAF ..... MAF; (d) Ad-12 ..... MAF ..... MAF; (e) Ad-2 ..... WI ..... KB; (f) Ad-12 ..... MAF ..... KB. Input multiplications were used identical to those in the first experiment. Electron microscopy Cells were trypsinized at various intervals of infection and centrifuged to form pellets. The pellets were fixed with Dalton's buffered osmium tetroxide (pH 7.6) for 1 hour. After dehydration through a series of ascending ethyl alcohol solutions, the specimens were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate..
Results and observations Non-infected and infected KB, WI, MAF cells by adenovirus type 2 and 12: Normal KB cells have been described (MARTINEZ-PALOMO et al. 1967). The normal WI and MAF cells contained large nuclei with homogeneous dispersal of chromatin and a prominent nucleolus. With the exception of Ad-12 .,.. WI, many intranuclear viral particles, dense patches, fibrils were often present in the viruses infected nuclei. The viral particles, in many cases, were in a dispersed form and in some cases were in crystalline (figs. 1 and 2). The average number of viral particles per nucleus and percentage of cells infected during maturation were listed in table 1. In the experiment of Ad-12 .,.. WI, no viral particles were found. Nuclei looked rather normal as compared with other preparations. However, some of the nuclei have been lyzed by the infection. KB cells infected with Ad-12, lattice structure with subunits are found (fig. 3).
Fig. 1. Portion of KB cell 44 hours after inoculation with adenovirus type 2. Viral particles (V) and dense bodies (D) are present in the nucleus. x 9,000. Fig. 2. Portion of a KB cell 44 hours ·after inoculation with adenovirus type 12. Electron dense bodies (p), patches, stippHngs and viral particles (V) are seen. x 13,500.
10
,.... ,....
WI MAF
AD-2 AD-2 AD-2 AD-12 AD-12 AD-12 AD-2 WI AD-2 WI AD-2 MAF AD-2 MAF AD-12 WI AD-12 WI AD-12 MAF AD-12 MAF
KB
MAF
KB
WI
KB
MAF
KB
WI
KB
WI MAF
KB
Cell line
Virus type
Table 1. Summary of the Results
42 40 96 14 35 32 87 37 69 21 32 22 36 15
No. observed nuclei
22 14 3 22 28 0 20 0 3' 0 9 0 5
11
No. nuclei with virus 26 55 15 30 63 87 0 54 0 14 0 41 0 33
% nuclei with virus
0 25 0 15 0 60
-
3 10 80 0
-
10 92
% mature nuclear inclusions
350
770
433
1,400
570 1,400
700 227 730
Average No. virus/section
Fig. 3. KB cell infected with adenovirus type 12, viral crystalline array (V), large electron dense inclusions (D) and small lattice with subunits (L) are shown. X 89,000.
Fig. 4.. Portion of a MAF cell super-exposed with virus infected MAF cells. Viral particles arc not present in the nucleus (N). x 9,000.
12
Fig. 5. Portion of a KB cell super-exposed with virus infected MAF cells. Electron-dense bodies (D), stipplings, viral crystalline arrays (V). Aggregates of microtubules (M:T) are present in the nucleus. x 45,000.
13
6
Fig. 6. Portion of a KB cell super-exposed with virus infected MAF cells to show several intranuclear crystalline structures. ER, endoplasmic reticulum; NE, nuclear envelope. x 50,000.
In the second passage, they were shown in table 1, none of those: Ad-2-WI-WI, Ad-12WI-WI, Ad-2-MAF-MAF, Ad-12-MAF-MAF were found to have viral production. Neither were there dense osmiophilic materials and nuclear inclusions (fig. 4). Fifty-four percent of the observed nuclei have well defined viral particles, either scattered throughout the entire nucleus or forming ordered arrays of viral particles. Nuclei are showing different degrees of lysis, at the sites with aggregates of viral particles. Ad-2 MAF _. KB Fourteen percent of the observed nuclei have produced viral particles which were found to be scattered throughout the nucleus. Dense osmiophilic materials were observed in most of the nuclei which show different degrees of lysis caused by the infectious viruses. A large proportion of the cells cultured on coverslip showed infection at different degrees. Ad-12 WI - KB The viral producing percentage is 410/0. Well defined viral particles were found to be mostly aggregated. Nuclear materials have been found to be displaced at where the viral aggregations are. Dense osmiophilic materials were found in most of the nuclei, and these nuclei were lysed at different degrees. Histological study has shown that many cells in this preparation have nuclear inclusions which indicates that the infection has taken place in most of the cells.
14
Ad-12 MAF - KB The viral particles, in a dispersed form or arrays were found in 33 % of the observed nuclei (fig. 5). Some of the nuclei show the presence of some polygonal crystals (fig. 6). These crystals are composed of two different sizes of microtubules (SUN 1971).
Discussion and conclusion No visible virions were found when infected WI cells with adenoviruses type 12 (Ad-12) although same dose of viruses has caused significant infection on MAF cells. The results obtained here could be interpreted as these: It has been known that the production of viral particles in nuclei, i. e., the replication of the viral DNA in nuclei, is due to a homology existing between the viral DNA and the DNA of the infected cell. The DNA of Ad-12 has a lower G-C percentage. There is probably no homology between the Ad-12 viral DNA and the DNA of WI cells. Therefore, no viral progeny can be produced in WI cells. It is interesting enough that none of these second passages (Ad-2 WI - WI, Ad-12 WI - WI; Ad-2 MAF - MAF, Ad-12 MAF - MAF) was found to have viral production. This indicates that, apparently, the infections on both WI and MAF cells caused by both Ad-2 and Ad-12 cannot be serially transmitted. However, when these infected cells were used to expose KB cells, significant viral yields were obtained. This indicates that the above mentioned cells might still carry the viral specific antigens although no visible virions were observed electron microscopically. The large protein crystals occurred in KB cells infected by adenovirus type 12 that had undergone passage once in MAF cells. WEVER and STICH (1969) found that one strain of adenovirus type 2 was passed in KB cells formed crystal structure in HEp2 cells. HENRY et al. (1971) also found crystals in Vero or Hela cell cultures infected with Ad-2. It may be suggested that the crystal formation appeared to be dependent on virus strain as well as on the host cell lines.
Literature HENRY, C. J., M. SLIFKIN, L. P. MERKOW and M. PARDO, The ultrastructure and nature of adenovirus type 2 induced paracrystalline formations. Virology 44, 215-218 (1971). MARTINEZ-PALOMO, A., J. LE BUIS and W. BERNHARD, Electron microscopy of adenovirus 12 replication. Fine structural changes in the nucleus of infected KB cells. J. Virol. 1, 817-829 (1967). PINKERTON, H., V. PALERMO, C. N. SUN and S. GOODMAN, Cytopathology in vitro of infection with adeno-viruses 12 and 2 in neoplastic and non-neoplastic cell lines of human and hamster origin. Lab. Investig. 11), 1123 (1966). SUN, C. N., Paracrystalline formation in cells infected by adenovirus type 12. Zbl. Bakt. Hyg. 219, 141 (1972). WEVER, J., and H. F. STICH, Electron microscopy of cell infected with adenovirus type 2. J. Virol. 3, 198-204 (1969). Author's address: C. N. SUN, Ph. D., EM Lab, Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, Arkansas 72206 (U.S.A.).
15
Human cell cultures infected by tumor-inducing and non-tumor-inducing viruses, an electron microscopic study By C. N.
SUN,
D. Hsu and H.
PINKERTON
With 6 figures (Received October 20, 1976) Key words: tumor-inducing viruses; non-tumor-inducing viruses; adenovirus; cell cultures: viral infection; virions; nuclear inclusions; human fibroblasts; viral specific antigens; electron microscopy
Summary This study is dealing with the infection patterns of adenovirus type 2 and type 12 on human fibroblast cell lines, KB, WI and MAF. With the exception of Ad-12 -+ WI, many intranuclear viral particles were present. None of these second passages (Ad-2 WI- WI, Ad-12 WI- WI, Ad-2 MAF-MAF, Ad-12 MAF-MAF) was found to have viral production. This indicates that the injections on both WI and MAF cells caused by both Ad-2 and Ad-12 cannot be serially transmitted. However, when these infected cells were used to expose KB cells, significant viral yields were obtained. This shows that the infected cells might still carry the viral specific antigens although no visible virions were observed. In regard to the hamster fibroblasts, the Ad-2 virus induces inclusions which show many intranuclear viral particles (as do other susceptible cells); the Ad-12 virus, on the other hand, only induces the early stages of nuclear inclusions, and no viral particles can be found (PINKERTON et al. 1966). Since it is obvious that type 12 adenovirus viral particles cannot be produced in the hamster cells, it is interesting to seek the differences between oncogenic (type 12) and nononcogenic (type 2) adenoviruses when they infect the human cells. The present research is dealing with the infection patterns of these two types of adenoviruses on two human fibroblast cell lines, WI and MAF. Comparative studies were made on the production of visible virions, the appearance of the mature nuclear inclusions, and all the abnormalities of the infected nuclei.
Material and methods Cell cultures and viruses Monolayers of three lines of human fibroblasts, KB, WI (originated from human-embryonic lung, Microbiological Associates, Bethesda, Maryland), MAF (derived from human skin and muscle fibroblasts) were cultured on plates with 1 x 106 cells per plate, cultures were transferred 3 or 4 days before infection was made, and were grown at 37°C in 5 ml of 2X Eagles' 10 % serum. All cell lines were infected separately with adenoviruses type 2 and type 12 (Ad-2 and Ad-12) at identical input multiplicities of 1-2 plaque forming units (PFU) per cell. Absorption of virus was carried out for 2 hours. After virus inoculum was removed, monolayers were washed twice overlayed with 2 % horse serum and incubated for 2 to 5 days, depending on the degree of cytopathology noted in the living cell monolayers. From each preparation, 2 plates were harvested and prepared for electron microscopy. Companion monolayers cultured on coverslips were infected in the same way for histo-cytological study. Coverslips were taken out of plates at the same intervals as were the plates pelleted for electron. microscopy, washed and removed the serum, fixed in absolute ethyl alcohol and stained with hematoxylin and eosin.
9
Second passages Second passages were made as follows: (a) Ad-2 ..... WI ..... WI. WI cells were infected with adenovirus type 2, which was propagated in WI line of cells. (b) Ad-12 ..... WI ..... WI. WI cells were infected with adenovirus type 12, which was propagated in WI line of cells. (c) Ad-2 ..... MAF ..... MAF; (d) Ad-12 ..... MAF ..... MAF; (e) Ad-2 ..... WI ..... KB; (f) Ad-12 ..... MAF ..... KB. Input multiplications were used identical to those in the first experiment. Electron microscopy Cells were trypsinized at various intervals of infection and centrifuged to form pellets. The pellets were fixed with Dalton's buffered osmium tetroxide (pH 7.6) for 1 hour. After dehydration through a series of ascending ethyl alcohol solutions, the specimens were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate..
Results and observations Non-infected and infected KB, WI, MAF cells by adenovirus type 2 and 12: Normal KB cells have been described (MARTINEZ-PALOMO et al. 1967). The normal WI and MAF cells contained large nuclei with homogeneous dispersal of chromatin and a prominent nucleolus. With the exception of Ad-12 .,.. WI, many intranuclear viral particles, dense patches, fibrils were often present in the viruses infected nuclei. The viral particles, in many cases, were in a dispersed form and in some cases were in crystalline (figs. 1 and 2). The average number of viral particles per nucleus and percentage of cells infected during maturation were listed in table 1. In the experiment of Ad-12 .,.. WI, no viral particles were found. Nuclei looked rather normal as compared with other preparations. However, some of the nuclei have been lyzed by the infection. KB cells infected with Ad-12, lattice structure with subunits are found (fig. 3).
Fig. 1. Portion of KB cell 44 hours after inoculation with adenovirus type 2. Viral particles (V) and dense bodies (D) are present in the nucleus. x 9,000. Fig. 2. Portion of a KB cell 44 hours ·after inoculation with adenovirus type 12. Electron dense bodies (p), patches, stippHngs and viral particles (V) are seen. x 13,500.
10
,.... ,....
WI MAF
AD-2 AD-2 AD-2 AD-12 AD-12 AD-12 AD-2 WI AD-2 WI AD-2 MAF AD-2 MAF AD-12 WI AD-12 WI AD-12 MAF AD-12 MAF
KB
MAF
KB
WI
KB
MAF
KB
WI
KB
WI MAF
KB
Cell line
Virus type
Table 1. Summary of the Results
42 40 96 14 35 32 87 37 69 21 32 22 36 15
No. observed nuclei
22 14 3 22 28 0 20 0 3' 0 9 0 5
11
No. nuclei with virus 26 55 15 30 63 87 0 54 0 14 0 41 0 33
% nuclei with virus
0 25 0 15 0 60
-
3 10 80 0
-
10 92
% mature nuclear inclusions
350
770
433
1,400
570 1,400
700 227 730
Average No. virus/section
Fig. 3. KB cell infected with adenovirus type 12, viral crystalline array (V), large electron dense inclusions (D) and small lattice with subunits (L) are shown. X 89,000.
Fig. 4.. Portion of a MAF cell super-exposed with virus infected MAF cells. Viral particles arc not present in the nucleus (N). x 9,000.
12
Fig. 5. Portion of a KB cell super-exposed with virus infected MAF cells. Electron-dense bodies (D), stipplings, viral crystalline arrays (V). Aggregates of microtubules (M:T) are present in the nucleus. x 45,000.
13
6
Fig. 6. Portion of a KB cell super-exposed with virus infected MAF cells to show several intranuclear crystalline structures. ER, endoplasmic reticulum; NE, nuclear envelope. x 50,000.
In the second passage, they were shown in table 1, none of those: Ad-2-WI-WI, Ad-12WI-WI, Ad-2-MAF-MAF, Ad-12-MAF-MAF were found to have viral production. Neither were there dense osmiophilic materials and nuclear inclusions (fig. 4). Fifty-four percent of the observed nuclei have well defined viral particles, either scattered throughout the entire nucleus or forming ordered arrays of viral particles. Nuclei are showing different degrees of lysis, at the sites with aggregates of viral particles. Ad-2 MAF _. KB Fourteen percent of the observed nuclei have produced viral particles which were found to be scattered throughout the nucleus. Dense osmiophilic materials were observed in most of the nuclei which show different degrees of lysis caused by the infectious viruses. A large proportion of the cells cultured on coverslip showed infection at different degrees. Ad-12 WI - KB The viral producing percentage is 410/0. Well defined viral particles were found to be mostly aggregated. Nuclear materials have been found to be displaced at where the viral aggregations are. Dense osmiophilic materials were found in most of the nuclei, and these nuclei were lysed at different degrees. Histological study has shown that many cells in this preparation have nuclear inclusions which indicates that the infection has taken place in most of the cells.
14
Ad-12 MAF - KB The viral particles, in a dispersed form or arrays were found in 33 % of the observed nuclei (fig. 5). Some of the nuclei show the presence of some polygonal crystals (fig. 6). These crystals are composed of two different sizes of microtubules (SUN 1971).
Discussion and conclusion No visible virions were found when infected WI cells with adenoviruses type 12 (Ad-12) although same dose of viruses has caused significant infection on MAF cells. The results obtained here could be interpreted as these: It has been known that the production of viral particles in nuclei, i. e., the replication of the viral DNA in nuclei, is due to a homology existing between the viral DNA and the DNA of the infected cell. The DNA of Ad-12 has a lower G-C percentage. There is probably no homology between the Ad-12 viral DNA and the DNA of WI cells. Therefore, no viral progeny can be produced in WI cells. It is interesting enough that none of these second passages (Ad-2 WI - WI, Ad-12 WI - WI; Ad-2 MAF - MAF, Ad-12 MAF - MAF) was found to have viral production. This indicates that, apparently, the infections on both WI and MAF cells caused by both Ad-2 and Ad-12 cannot be serially transmitted. However, when these infected cells were used to expose KB cells, significant viral yields were obtained. This indicates that the above mentioned cells might still carry the viral specific antigens although no visible virions were observed electron microscopically. The large protein crystals occurred in KB cells infected by adenovirus type 12 that had undergone passage once in MAF cells. WEVER and STICH (1969) found that one strain of adenovirus type 2 was passed in KB cells formed crystal structure in HEp2 cells. HENRY et al. (1971) also found crystals in Vero or Hela cell cultures infected with Ad-2. It may be suggested that the crystal formation appeared to be dependent on virus strain as well as on the host cell lines.
Literature HENRY, C. J., M. SLIFKIN, L. P. MERKOW and M. PARDO, The ultrastructure and nature of adenovirus type 2 induced paracrystalline formations. Virology 44, 215-218 (1971). MARTINEZ-PALOMO, A., J. LE BUIS and W. BERNHARD, Electron microscopy of adenovirus 12 replication. Fine structural changes in the nucleus of infected KB cells. J. Virol. 1, 817-829 (1967). PINKERTON, H., V. PALERMO, C. N. SUN and S. GOODMAN, Cytopathology in vitro of infection with adeno-viruses 12 and 2 in neoplastic and non-neoplastic cell lines of human and hamster origin. Lab. Investig. 11), 1123 (1966). SUN, C. N., Paracrystalline formation in cells infected by adenovirus type 12. Zbl. Bakt. Hyg. 219, 141 (1972). WEVER, J., and H. F. STICH, Electron microscopy of cell infected with adenovirus type 2. J. Virol. 3, 198-204 (1969). Author's address: C. N. SUN, Ph. D., EM Lab, Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, Arkansas 72206 (U.S.A.).
15
