Intervirology 5: 354-363 (1975)
Cytomégalovirus Infection of Human Lung Epithelial Cells in vitro Susan M ichelson-F iske', J acqueline A rnoult and H enri F ebvre Unité de Biologie Cellulaire U 47 de l'Institut National de la Santé et de la Recherche Médicale, Paris
Key Words. Cytomegalovirus infection • Epithelial cells Summary. Human lung epithelial cells were productively infected with human cyto megalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.
Cytomegalovirus (CMV), a member of the herpes group, is supposedly both markedly host- and tissue-specific in vitro. The in vitro host specificity of CMV has recently been questioned by Plummer and G oodheart [I ] and seems to be less pronounced than originally thought. Heterologous infections have been performed [1-7], though many such infections are transitory and/or produce no infectious particles. The in vitro tissue specificity of human CMV is paradoxical. This virus is mostly found in vivo in epithelial cells, whereas in vitro it so far seems that epithelial cells are insusceptible, unless one resorts to chemical induction. In seeking a human cell ‘nonpermissive’ for CMV in which to study an abortive cycle, we first tested human lung epithelium as a possible in vitro system. We would like to report briefly on a successful infection in vitro of these human lung epithelial cells with an isolate of human CMV (Mira strain).2
Received: August 5, 1975.
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1 Presen (address: Laboratoire de Médecine expérimentale du Collège de France, 11, place Marcelin-Berthclol, F-75231 Paris Cédex 05 (France). 2 Generous gift of Dr. A ndré Boüé, Centre International de l'Enfance. Paris, who iso lated this virus from a newborn with generalized disease.
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Fi f I . a Phase contrast of lung epithelial cells, x 180. h Lung fibroblasts from the same culture, x j80. c 'Lamellar bodies’ formed in the lung epithelial cells, x 27,000.
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Materials and Methods Cultures. Cell cultures were grown in 1640 medium supplemented with 8% fetal calf serum, in 75-cm2 Falcon flasks in an atmosphere of 5% CO2 and air. No other antibiotics except 3% tylosine were used. Small fragments of lung from a 6'/2-month-old human embryo were grown in Falcon plastic culture flasks. The cultures were periodically cleared of fibroblasts by trypsinization (about 2-5 min in 0.25% trypsin in PBS buffer at 37 ) which did not affect the epithelial cells. The fibroblasts thus recovered were grown out into a cell strain and used for titration of the virus. Infection. Six weeks after the initiation of the cultures, the cells were trypsinized to remove a maximum of fibroblasts, and infected with 5.3 x 10® PFU of the Mira isolate in 3 ml of 1640 medium without serum. After an adsorption of I h at 37 , 15 ml of 1640 medium + 8% fetal calf serum were added. The medium was thenceforth changed every 2-4 days. Microscopy. Samples for the electron microscope were prepared by fixation of a flask at 2, 4 and 8 weeks after infection. Cultures were fixed in 2.5% glutaraldehyde in PBS buffer (I h) followed by 2% OsOt in veronal buffer (I h) and were embedded in situ in a mixture of araldite and Epon.
Results
Fig.2. a Mononucleated lung epithelial-type cell, x 6,000. b Multinucleated lung epi thelial-type cell, x 4,200. c Lung fibroblast from the same culture. Note the absence of fibrils and the characteristic fusiform, x 16,800.
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Before infection, the cultures consisted of a monolayer of epithelioid cells (fig. la) extending from the lung explants. Fibroblasts (fig. lb) were seen in areas between plaques of epithelioid cells. The growth of the epithelioid cells was very slow. All attempts to passage them, either before or after infection, failed. In contrast, the fibroblasts trypsinized from these flasks before infection have given rise to an abundant cell strain which has so far undergone 20 passages with no spontaneous alterations. Two weeks after infection, the infected fibroblasts had died off and only the epithelioid cells and a few lung explants remained, both of which showed no evidence of infection by light microscopy. Electron microscopy revealed, however, that infection had occurred in cells that did not have the characteristics of fibroblasts. No fibroblasts were seen (fig. Ic). Two kinds of cells were present: (1) Multinucleated cells with up to 12 nuclei in one cytoplasm. The nuclei were pale and often contained punctiform, scattered densifications as well
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as occasional clusters of perichromatin granules. In addition to some microfilaments, the cytoplasm contained characteristic bodies delimited by a mem brane and composed of layers of dense myelin-like substance arranged either concentrically or in parallel bars (fig. Ic). The cells were joined to one another by an intertwining of the microvillosities of their cell membranes and oc casional desmosomes (fig. 2b). (2) Large, mononucleated cells with a striking number of bundles of fibrils and which were joined to one another by desmosomes and intertwining cell membranes (fig. 2a). At 2 weeks after infection only a few cells presented the typical lesions of CM V infection. At 4 weeks the characteristic features of CMV infection were observed in all cells, while the cells retained their original epithelial aspect (fig. 3a, b). The nuclei were enlarged with a light cytoplasm, contained the charac teristic inclusion body (skein: fig.4a), and all nuclear stages of maturation of virus were observed. There was no margination of chromatin. In the cyto plasm the Golgi zone was strikingly augmented with many multivesicular bodies around which virus had accumulated (fig.4b). However, dense bodies usually found in CMV infection were very rare.
Since it was isolated and propagated in vitro [8], the replication of human CMV previously seemed to be restricted to human fibroblastic tissue. The fibroblast tropism of CMV in vitro is mentioned in numerous reviews [9-12] and has even served as an identifying criterion in the isolation of this virus [13] and in studies to determine its presence in circulating leukocytes [14], Here we show that at least one type of human lung epithelial cell can be productively infected by CMV in vitro. Evidence that these are epithelial cells rests on their typical morphology, on the one hand, and on their behavior in vitro, on the other. Morphologically, these cells bear features of pneumocyte type II or corner cells of the lung. The identification at the ultrastructural level rests not only on the interconnection of cells (intertwining of surface membranes with occasional desmosomes) and on the abundance of micro fibrils in the ground cytoplasm, but also on the presence of'lamellar bodies’ characteristically found in this type of cell [15,16], Furthermore, the cells are multinucleated and it is well known that type II pneumocytes form syncytia upon infection with viruses.
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Discussion
359
Fig.3. a Two CMV-infected multinucleated epithelial cells, x 8,000. b CMV-infected mononucleated epithelioid cell, x 8,000. In both cell types note the characteristic reticu lar inclusion bodies in the nuclei and the virus accumuiation in the cytoplasm in multivesicular bodies.
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Cytomegalovirus Infection of Human Lung Epithelial Cells in vitro
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Fig.4. a Fligh magnification of a nuclear inclusion body (skein) showing associated virus in various stages of maturation, x 20,000. b A multivesicuiar body in the cytoplasm of an infected epithelioid cell. Note the presence of enveloped virus and particles forming in the surrounding cytoplasm, x 21,000.
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The epithelial nature of the second type of cell is less certain. Although they are rich in microfibrils and joined to one another by desmosomes or intertwining cell membranes, thereby strongly resembling epithelial cells of which they might represent another aspect, they still might correspond to pleural (mésothélial) cells which also display epithelioid features. Whether this second type of cell be considered mésothélial or not, its characteristics of growth and passage ability are like those of the first type of cell in that its rate of multiplication is almost nil and it cannot be passaged. That the infection is caused by CM V and not by another herpes-type virus which might have contaminated the preparation rests on the really unique morphological characteristics induced by CMV. As pointed out by others [ 17] and by ourselves [18, ref. : 20], it is possible to distinguish the lesions caused by CMV at the ultrastructural level on the basis of the absence of margination of chromatin and the reticular aspect of the inclusion bodies not found with other herpesviruses. However, one characteristic feature of CMV lesions, the presence of dense bodies, is rarely found. It still has to be demonstrated, therefore, that a modification of the virus has not occurred which might also account for its ability to infect epithelial cells. This is unlikely, however, since in the few remaining fibroblasts seen in the 2-week preparation, lysosomelike bodies were observed ‘en masse’. The supernatant of such cultures 8 weeks after infection also provoked typical cytopathic effects when inoculated into lung fibroblasts from the same embryo, and destroyed such cultures within 8 days. A possible relationship between the rarity of lysosome-Iike bodies and nonlytic CMV infection has been suggested by R uebner et al. [19] in their study of the persistence of murine CMV in salivary glands of mice following intra peritoneal infection of the virus. Thus, epithelial cells might be a useful tool for the in vitro study of the role of lysosome-like bodies in lytic infection with this virus. In conclusion, it may be said that, contrary to the accepted notion, human lung epithelial cells may be productively infected with CMV in vitro and that they support viral replication without, however, manifesting cytopathogenic effects or undergoing cell lysis. Contrary to our expectations, this study shows that epithelial cells are a poor candidate for the study of abortive CMV in-
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Cytomegalovirus Infection of Human Lung Epithelial Cells in vitro 361
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fection, but it does show the possible importance of epithelial cells in investi gating the steady-state nature of CMV infection as it exists in vivo, where it is expressed by long-term excretion of virus in urine or saliva. The steadystate nature of this infection contrasts with the integrated type of infection of heterologous systems where cytopathogenic effects arc observed but no infectious virus is produced [6].
A cknowledgments We wish to thank Dr. H aguenau and Dr. Basset for their advice in the preparation of this text and Mrs. Picart for her expert technical assistance.
1 P lummer, G. and G oodheart, C. R .: Growth of murine cytomegalovirus in a hetero logous cell system and its enhancement by 5-iododeoxyuridine. Infec. Immunity 10: 251-256(1974). 2 K im, K.S. and C arp, R. I .: Abortive infections of human diploid cells by murine cyto megalovirus. Infec. Immunity 6: 793-797 (1972). 3 F ioretti, A . ; F urukawa , T . : S antoli, D., and P lotkin, S . : Non-productive infection of guinea pig cells with human cytomegalovirus. J. Virol. 11: 998-1003 (1973). 4 A lbrecht, T. and R app , F.: Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55:53-61 (1973) . 5 W anes, J.L. and W eller, T. H.: Behavior of human cytomegalovirus in cell cultures of bovine and simian origin. Proc.Soc. exp. Biol. Med. 145: 379-384(1974). 6 G onczol, E.; Boldogh, I.; Vaczi, L., and G ergely, L.: The expression of virus genome in mouse cells inoculated by a human strain of cytomegalovirus (HCMV). 9th Meet. Eur. Tumor Virus Group, Mariehamn, Aland 1975. 7 St . J eor, S. and R app , F .: CMV conversion of nonpermissive cells to a permissive state for viral replication. Science, N.Y. 181: 1060-1061 (1973). 8 Smith, M.G.: Propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus disease. Proc. Soc. exp. Biol. Med. 92: 424-430 ( 1965). 9 H anshaw , J.B.: Cytomegaloviruses; in Virology Monographs, No.3, pp. 1-23 (Sprin ger, New York 1968). 10 W eller, T.H .: The cytomegaloviruses: ubiquitous agents with protean clinical mani festations. New Engl. J. Med. 285: 203-214, 267-274 (1971). 11 W right , H.T.: Cytomegaloviruses; in K aplan The Herpesviruses, chap. 12, pp.353388 (Academic Press, New York 1973). 12 R app , F.: Herpesviruses and cancer; in Advances in cancer research, vol. 19, pp. 265-302 (1974) . 13 R owe , W.P.; Hartley, J.W .; W aterman, S.; T urner, H.C., and H uebner, R.J.: Cytopathogenic agents resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc. Soc. exp. Biol. Med. 92:418-424 (1956).
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References
Cytomegalovirus Infection of Human Lung Epithelial Cells in vitro
363
14 Lang, D. J. and N oren, B.: Cytomegaloviremia following congenital infection. J. Pediat. 73: 812-819 (1968). 15 C ampiche, M .A.; G autier, A.; H ernandez, E.I., and R eymond, A.: An electron microscope study of the development of human lung. Pediatrics, Springfield 32:976-994 (1963). 16 Basset, F.: Notions d’ultrastructurc pulmonaire. Presse med. 77: 123-127 (1969). 17 S mith, J.D . and de H arven, E.: Herpes simplex and human cytomegalovirus replication in W1 38 cells. 1. Sequence of viral replication. J. Virol. 12: 919-930 (1973). 18 G iraldo, G .; Beth, E., and H aguenau, F r .: Presence of herpes type virus particles in tissue culture of 5 cases of Kaposi's sarcoma from different geographical regions. A light and electron microscopical study. J. natn.Cancer Inst. 49: 1509-1526 (1972). 19 R uebner, B.H.; H irano , T.: S lusser, R.; O sborn, J., and M edearis, D.N., jr .: Cyto megalovirus infection: viral ultrastructurc with particular reference to the relationship of lysosomes to cytoplasmic inclusions. Am. J. Path. 48: 971-989 (1966). 20 H aguenau, F r . and M ichelson-F iske, S.: Cytomegalovirus: nucleocapsid assembly and core structure. Intervirology 5: 293-299 (1975).
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Dr. S usan M icheeson-F iske, Laboratoire de Médecine expérimentale, Collège de France, 11, place Marcelin-Berthelot, F-75231 Paris Cédex 05 (France)
Cytomégalovirus Infection of Human Lung Epithelial Cells in vitro Susan M ichelson-F iske', J acqueline A rnoult and H enri F ebvre Unité de Biologie Cellulaire U 47 de l'Institut National de la Santé et de la Recherche Médicale, Paris
Key Words. Cytomegalovirus infection • Epithelial cells Summary. Human lung epithelial cells were productively infected with human cyto megalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.
Cytomegalovirus (CMV), a member of the herpes group, is supposedly both markedly host- and tissue-specific in vitro. The in vitro host specificity of CMV has recently been questioned by Plummer and G oodheart [I ] and seems to be less pronounced than originally thought. Heterologous infections have been performed [1-7], though many such infections are transitory and/or produce no infectious particles. The in vitro tissue specificity of human CMV is paradoxical. This virus is mostly found in vivo in epithelial cells, whereas in vitro it so far seems that epithelial cells are insusceptible, unless one resorts to chemical induction. In seeking a human cell ‘nonpermissive’ for CMV in which to study an abortive cycle, we first tested human lung epithelium as a possible in vitro system. We would like to report briefly on a successful infection in vitro of these human lung epithelial cells with an isolate of human CMV (Mira strain).2
Received: August 5, 1975.
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1 Presen (address: Laboratoire de Médecine expérimentale du Collège de France, 11, place Marcelin-Berthclol, F-75231 Paris Cédex 05 (France). 2 Generous gift of Dr. A ndré Boüé, Centre International de l'Enfance. Paris, who iso lated this virus from a newborn with generalized disease.
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355
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Fi f I . a Phase contrast of lung epithelial cells, x 180. h Lung fibroblasts from the same culture, x j80. c 'Lamellar bodies’ formed in the lung epithelial cells, x 27,000.
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Materials and Methods Cultures. Cell cultures were grown in 1640 medium supplemented with 8% fetal calf serum, in 75-cm2 Falcon flasks in an atmosphere of 5% CO2 and air. No other antibiotics except 3% tylosine were used. Small fragments of lung from a 6'/2-month-old human embryo were grown in Falcon plastic culture flasks. The cultures were periodically cleared of fibroblasts by trypsinization (about 2-5 min in 0.25% trypsin in PBS buffer at 37 ) which did not affect the epithelial cells. The fibroblasts thus recovered were grown out into a cell strain and used for titration of the virus. Infection. Six weeks after the initiation of the cultures, the cells were trypsinized to remove a maximum of fibroblasts, and infected with 5.3 x 10® PFU of the Mira isolate in 3 ml of 1640 medium without serum. After an adsorption of I h at 37 , 15 ml of 1640 medium + 8% fetal calf serum were added. The medium was thenceforth changed every 2-4 days. Microscopy. Samples for the electron microscope were prepared by fixation of a flask at 2, 4 and 8 weeks after infection. Cultures were fixed in 2.5% glutaraldehyde in PBS buffer (I h) followed by 2% OsOt in veronal buffer (I h) and were embedded in situ in a mixture of araldite and Epon.
Results
Fig.2. a Mononucleated lung epithelial-type cell, x 6,000. b Multinucleated lung epi thelial-type cell, x 4,200. c Lung fibroblast from the same culture. Note the absence of fibrils and the characteristic fusiform, x 16,800.
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Before infection, the cultures consisted of a monolayer of epithelioid cells (fig. la) extending from the lung explants. Fibroblasts (fig. lb) were seen in areas between plaques of epithelioid cells. The growth of the epithelioid cells was very slow. All attempts to passage them, either before or after infection, failed. In contrast, the fibroblasts trypsinized from these flasks before infection have given rise to an abundant cell strain which has so far undergone 20 passages with no spontaneous alterations. Two weeks after infection, the infected fibroblasts had died off and only the epithelioid cells and a few lung explants remained, both of which showed no evidence of infection by light microscopy. Electron microscopy revealed, however, that infection had occurred in cells that did not have the characteristics of fibroblasts. No fibroblasts were seen (fig. Ic). Two kinds of cells were present: (1) Multinucleated cells with up to 12 nuclei in one cytoplasm. The nuclei were pale and often contained punctiform, scattered densifications as well
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as occasional clusters of perichromatin granules. In addition to some microfilaments, the cytoplasm contained characteristic bodies delimited by a mem brane and composed of layers of dense myelin-like substance arranged either concentrically or in parallel bars (fig. Ic). The cells were joined to one another by an intertwining of the microvillosities of their cell membranes and oc casional desmosomes (fig. 2b). (2) Large, mononucleated cells with a striking number of bundles of fibrils and which were joined to one another by desmosomes and intertwining cell membranes (fig. 2a). At 2 weeks after infection only a few cells presented the typical lesions of CM V infection. At 4 weeks the characteristic features of CMV infection were observed in all cells, while the cells retained their original epithelial aspect (fig. 3a, b). The nuclei were enlarged with a light cytoplasm, contained the charac teristic inclusion body (skein: fig.4a), and all nuclear stages of maturation of virus were observed. There was no margination of chromatin. In the cyto plasm the Golgi zone was strikingly augmented with many multivesicular bodies around which virus had accumulated (fig.4b). However, dense bodies usually found in CMV infection were very rare.
Since it was isolated and propagated in vitro [8], the replication of human CMV previously seemed to be restricted to human fibroblastic tissue. The fibroblast tropism of CMV in vitro is mentioned in numerous reviews [9-12] and has even served as an identifying criterion in the isolation of this virus [13] and in studies to determine its presence in circulating leukocytes [14], Here we show that at least one type of human lung epithelial cell can be productively infected by CMV in vitro. Evidence that these are epithelial cells rests on their typical morphology, on the one hand, and on their behavior in vitro, on the other. Morphologically, these cells bear features of pneumocyte type II or corner cells of the lung. The identification at the ultrastructural level rests not only on the interconnection of cells (intertwining of surface membranes with occasional desmosomes) and on the abundance of micro fibrils in the ground cytoplasm, but also on the presence of'lamellar bodies’ characteristically found in this type of cell [15,16], Furthermore, the cells are multinucleated and it is well known that type II pneumocytes form syncytia upon infection with viruses.
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Discussion
359
Fig.3. a Two CMV-infected multinucleated epithelial cells, x 8,000. b CMV-infected mononucleated epithelioid cell, x 8,000. In both cell types note the characteristic reticu lar inclusion bodies in the nuclei and the virus accumuiation in the cytoplasm in multivesicular bodies.
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Cytomegalovirus Infection of Human Lung Epithelial Cells in vitro
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Fig.4. a Fligh magnification of a nuclear inclusion body (skein) showing associated virus in various stages of maturation, x 20,000. b A multivesicuiar body in the cytoplasm of an infected epithelioid cell. Note the presence of enveloped virus and particles forming in the surrounding cytoplasm, x 21,000.
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The epithelial nature of the second type of cell is less certain. Although they are rich in microfibrils and joined to one another by desmosomes or intertwining cell membranes, thereby strongly resembling epithelial cells of which they might represent another aspect, they still might correspond to pleural (mésothélial) cells which also display epithelioid features. Whether this second type of cell be considered mésothélial or not, its characteristics of growth and passage ability are like those of the first type of cell in that its rate of multiplication is almost nil and it cannot be passaged. That the infection is caused by CM V and not by another herpes-type virus which might have contaminated the preparation rests on the really unique morphological characteristics induced by CMV. As pointed out by others [ 17] and by ourselves [18, ref. : 20], it is possible to distinguish the lesions caused by CMV at the ultrastructural level on the basis of the absence of margination of chromatin and the reticular aspect of the inclusion bodies not found with other herpesviruses. However, one characteristic feature of CMV lesions, the presence of dense bodies, is rarely found. It still has to be demonstrated, therefore, that a modification of the virus has not occurred which might also account for its ability to infect epithelial cells. This is unlikely, however, since in the few remaining fibroblasts seen in the 2-week preparation, lysosomelike bodies were observed ‘en masse’. The supernatant of such cultures 8 weeks after infection also provoked typical cytopathic effects when inoculated into lung fibroblasts from the same embryo, and destroyed such cultures within 8 days. A possible relationship between the rarity of lysosome-Iike bodies and nonlytic CMV infection has been suggested by R uebner et al. [19] in their study of the persistence of murine CMV in salivary glands of mice following intra peritoneal infection of the virus. Thus, epithelial cells might be a useful tool for the in vitro study of the role of lysosome-like bodies in lytic infection with this virus. In conclusion, it may be said that, contrary to the accepted notion, human lung epithelial cells may be productively infected with CMV in vitro and that they support viral replication without, however, manifesting cytopathogenic effects or undergoing cell lysis. Contrary to our expectations, this study shows that epithelial cells are a poor candidate for the study of abortive CMV in-
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fection, but it does show the possible importance of epithelial cells in investi gating the steady-state nature of CMV infection as it exists in vivo, where it is expressed by long-term excretion of virus in urine or saliva. The steadystate nature of this infection contrasts with the integrated type of infection of heterologous systems where cytopathogenic effects arc observed but no infectious virus is produced [6].
A cknowledgments We wish to thank Dr. H aguenau and Dr. Basset for their advice in the preparation of this text and Mrs. Picart for her expert technical assistance.
1 P lummer, G. and G oodheart, C. R .: Growth of murine cytomegalovirus in a hetero logous cell system and its enhancement by 5-iododeoxyuridine. Infec. Immunity 10: 251-256(1974). 2 K im, K.S. and C arp, R. I .: Abortive infections of human diploid cells by murine cyto megalovirus. Infec. Immunity 6: 793-797 (1972). 3 F ioretti, A . ; F urukawa , T . : S antoli, D., and P lotkin, S . : Non-productive infection of guinea pig cells with human cytomegalovirus. J. Virol. 11: 998-1003 (1973). 4 A lbrecht, T. and R app , F.: Malignant transformation of hamster embryo fibroblasts following exposure to ultraviolet-irradiated human cytomegalovirus. Virology 55:53-61 (1973) . 5 W anes, J.L. and W eller, T. H.: Behavior of human cytomegalovirus in cell cultures of bovine and simian origin. Proc.Soc. exp. Biol. Med. 145: 379-384(1974). 6 G onczol, E.; Boldogh, I.; Vaczi, L., and G ergely, L.: The expression of virus genome in mouse cells inoculated by a human strain of cytomegalovirus (HCMV). 9th Meet. Eur. Tumor Virus Group, Mariehamn, Aland 1975. 7 St . J eor, S. and R app , F .: CMV conversion of nonpermissive cells to a permissive state for viral replication. Science, N.Y. 181: 1060-1061 (1973). 8 Smith, M.G.: Propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus disease. Proc. Soc. exp. Biol. Med. 92: 424-430 ( 1965). 9 H anshaw , J.B.: Cytomegaloviruses; in Virology Monographs, No.3, pp. 1-23 (Sprin ger, New York 1968). 10 W eller, T.H .: The cytomegaloviruses: ubiquitous agents with protean clinical mani festations. New Engl. J. Med. 285: 203-214, 267-274 (1971). 11 W right , H.T.: Cytomegaloviruses; in K aplan The Herpesviruses, chap. 12, pp.353388 (Academic Press, New York 1973). 12 R app , F.: Herpesviruses and cancer; in Advances in cancer research, vol. 19, pp. 265-302 (1974) . 13 R owe , W.P.; Hartley, J.W .; W aterman, S.; T urner, H.C., and H uebner, R.J.: Cytopathogenic agents resembling human salivary gland virus recovered from tissue cultures of human adenoids. Proc. Soc. exp. Biol. Med. 92:418-424 (1956).
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References
Cytomegalovirus Infection of Human Lung Epithelial Cells in vitro
363
14 Lang, D. J. and N oren, B.: Cytomegaloviremia following congenital infection. J. Pediat. 73: 812-819 (1968). 15 C ampiche, M .A.; G autier, A.; H ernandez, E.I., and R eymond, A.: An electron microscope study of the development of human lung. Pediatrics, Springfield 32:976-994 (1963). 16 Basset, F.: Notions d’ultrastructurc pulmonaire. Presse med. 77: 123-127 (1969). 17 S mith, J.D . and de H arven, E.: Herpes simplex and human cytomegalovirus replication in W1 38 cells. 1. Sequence of viral replication. J. Virol. 12: 919-930 (1973). 18 G iraldo, G .; Beth, E., and H aguenau, F r .: Presence of herpes type virus particles in tissue culture of 5 cases of Kaposi's sarcoma from different geographical regions. A light and electron microscopical study. J. natn.Cancer Inst. 49: 1509-1526 (1972). 19 R uebner, B.H.; H irano , T.: S lusser, R.; O sborn, J., and M edearis, D.N., jr .: Cyto megalovirus infection: viral ultrastructurc with particular reference to the relationship of lysosomes to cytoplasmic inclusions. Am. J. Path. 48: 971-989 (1966). 20 H aguenau, F r . and M ichelson-F iske, S.: Cytomegalovirus: nucleocapsid assembly and core structure. Intervirology 5: 293-299 (1975).
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Dr. S usan M icheeson-F iske, Laboratoire de Médecine expérimentale, Collège de France, 11, place Marcelin-Berthelot, F-75231 Paris Cédex 05 (France)
