Multiplication of Measles Virus in Cell Cultures

Transcription

1 BACTERIOLOGICAL REVIEWS, Mar., American Society for Microbiology Vol. 30, No. I Printed in U.S.A. Multiplication of Measles Virus in Cell Cultures MINORU MATUMOTO Institute for Infectious Diseases, University of Tokyo, Minato-ku, Tokyo, Japan INTRODUCTION CELL CULTURES FOR PRtMARy VIRUS ISOLATION CELL SUSC IBILrY RANGE IN CULTURE Primary Human Cell Cultures Continuous Cell Lines ofhuman Origin Monkey Cell Cultures Cell Cultures ofnonhuman and Nonsimian Origin CYTOPATHiC Emcrs General Observations Syncytial Formation Factors Affecting Cytopathogenesis Inclusion Bodies INFEcrIvrrY TITRATION AND PLAQUE FORMATION PRODUCTION OF ANTIGENS IN INFECTED CELL CULTURE ELECTRON MICROSCOPY OF INFECrED CELLS QUANTITATIVE ANALYSIS OF VIRUS GROWTH Growth Curve Latent Period Intracellular Multiplication of Virus Release of Intracellularly Produced Virus EFFECTS OF ACTINOMYCN D ON REPLICATION OF VIRus INTERFERON IN REPLICATION OF VIRUS CARRIER STATE CULTURE SUMMARY AND FUTURE OUTLOOK LITERATURE CITED INTRODUCTION Measles has been recognized as a clinical entity for many centuries, and the viral etiology was established almost a half century ago. However, until recently the investigation of measles had been greatly hampered by the lack of adequate means for consistent propagation of this agent in the laboratory. In 1954 this difficulty was overcome by Enders and Peebles (30), who showed that measles virus could be propagated readily in human and monkey cell cultures. Their study gave impetus to systematic investigations of the nature and behavior of measles virus as well as of the natural history and control measures of the disease. Since consistent work with measles virus became feasible, there have been several reviews (5, 13, 15, 32, 34, 66, 78, 141). The purpose of this review is to assemble data on the multiplication of measles virus in cell cultures, and to try to elucidate the mechanism of multiplication of this virus. CELL CULTURES FOR PRIMARY VIRUS ISOLATION Enders and Peebles (30) reported in 1954 that measles virus could be isolated in human and simian cell cultures from materials of measles 152 patients at an early stage of the disease. In these systems, multiplication of the virus was accompanied by characteristic cytopathic changes. These changes consisted of formation of multinucleated giant cells, vacuolization, and slow destruction of the whole cell sheet. Cytoplasmic and intranuclear eosinophilic inclusion bodies were seen in stained cultures. The cytopathic changes could be prevented by antibody, which during conva- appears in the serum of patients lescence, a fact indicating that these cytopathic effects were induced by the virus. Specific complement-fixing antigen was also demonstrated in the infected cultures. These findings have provided laboratory tools whereby the presence and antigenic effects of measles virus can be recognized easily and with complete assurance. Subsequent experience of various workers has indicated that isolation of measles virus can be achieved in primary cultures of human and simian cells. Human renal cell culture is most sensitive for primary isolation of the virus (30, 32, 33, 36, 47, 57, , 116, 127). Since human kidney is often difficult to obtain, primary cultures of human amnion, which can be obtained more easily, have been tried for this purpose, but have

2 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES been found to be less sensitive than primary cultures of human kidney. Thus, the findings of Ruckle (111) and Bech (9), who employed both human amnion and renal cell cultures, showed that, although the virus could be occasionally isolated in human amnion cell cultures, this occurred less frequently than in renal cell cultures. Wright (146) reported successful isolation in human amnion and chorion cultures, but gave no quantitative information. Zhdanov and his associates also isolated measles virus in human amnion cultures (149). Enders (36) reported that isolation attempts in human amnion cell cultures in his laboratory had rarely succeeded, as illustrated by the results obtained by Katz who isolated the virus only twice in 43 trials. Ruckle (113) isolated measles virus by culturing tissues obtained from a 21-month-old girl who died with severe central nervous system symptoms but no evidence of rash. The virus was recovered by this procedure from tissues of the lung, kidney, spleen, and lymph node, which exhibited giant cells in stained sections, but not from brain tissue. On the other hand, lung tissue was the only material from which the virus was isolated when tissue extracts were inoculated into primary cultures of human amnion. These findings suggest the advantage of revealing the presence of virus by use of the tissues rather than an extract as inoculum. Monkey kidney cell culture has been shown to be as sensitive as human renal cell culture for primary isolation of measles virus (8, 9, 22, 30, 32, 33, 73, 85, 111, 112, 114, 116, 147). Kidney tissues of rhesus monkey (8, 9, 30, 32, 111, 112), cynomolgus monkey (8, 9, 111, 112), baboon (8, 9), Macacafiuscata (73, 85), and Macaca cyclopis (73, 85) were all successfully used for primary isolation. Although monkey kidney cell culture is most sensitive to measles virus from specimens, the difficulties involved in using this culture for primary isolation are stressed, since monkey kidneys have been found to harbor measles virus occasionally (114, 115) and frequently the foamy agent and other simian viruses (8, 9, 22, 30, 73, 85, 111, 114, 117). Only a few trials have been made of primary virus isolation in primary tissue cultures of other animals. Frankel et al. (38) reported successful isolation of the virus in dog kidney cultures. As for continuous cell lines, most investigators have failed to isolate the virus in such systems (36). Frankel and West (39), however, reported success in the FL line of human amnion cells, without giving any details. Toyoshima et al. (132) also succeeded in isolating two strains in this cell culture from a total of 19 sera and throat washings. Yasui (148) also isolated a strain in FL cells. 153 Bech and von Magnus (8), using HeLa cells, failed to isolate the virus. However, Shingu and Nakagawa (126) isolated in HeLa cell cultures three strains from five blood specimens and none from two throat swabs. In spite of these positive results, it would seem justifiable to conclude that, at present, continuous cell lines are unsuitable for primary isolation, although they may be highly susceptible to tissue culture-adapted virus. In summary, it can be said that human and monkey kidney cell cultures are most sensitive for primary isolation of measles virus. These isolation methods, however, have drawbacks, since human kidney is often difficult to obtain, and monkey kidney frequently harbors latent agents, even measles virus occasionally. Hence, further investigations are necessary to find a more appropriate host for primary isolation of measles virus. CELL SUSCEPrIBILrY RANGE in CuLTruRE Once isolated in primary cultures of human or simian origin, measles virus is readily passaged in these cultures. With the strains thus established, little difficulty has been encountered in adapting the virus to a variety of continuous human or simian cell lines, originating from both normal and malignant cells. Adaptation of measles virus to cell cultures of certain other animal species has been accomplished with varying degrees of success. Primary Human Cell Cultures The primary human cell cultures thus far tested and found to be susceptible to measles virus include those of kidney (8, 9, 20, 30, 33, 37, , 147), amnion (8, 9, 32, 33, 82, , 147), chorion (146, 147), lung (113, 116), spleen (113), and lymph node (113) (Table 1). Berg and Rosenthal (11) showed that measles virus multiplied rapidly in suspensions of blood leukocytes. Monocytic elements were definitely involved, although the possibility of viral proliferation in polymorphonuclear cells could not be ruled out. Adaptation of the virus to primary human amnion cultures meets with little difficulty, although, as mentioned earlier, this host system is less sensitive to primary passage virus (82). Milovanovic and co-workers (82) found that establishment of a monkey tissue culture passage virus in amnion cultures required a period of adaptation lasting for several passages, during which large virus inocula were required and the cytopathic effect was either absent or very difficult to detect. Once the virus was adapted to the amnion culture, it did not lose its capacity to multiply in kidney cultures, and comparable infectivity titers were attained.

3 154 MATUMOTO TABLE 1. Susceptibility to measles virus of cultured cells Cell Suscep- Reference tibility Human (primary culture) Kidney Amnion Chorion Lung Spleen Lymph node Leukocyte Human (stable line) Kidney (Chang) Lung Lung (Lu106) Liver (Chang) Amnion (FL) Amnion (JTC3) Conjunctiva (Chang) Intestinal mucosa (Henle and Deinhardt) Nasal mucosa (DMB, DHov) Bone marrow (Detroit 6) Bone marrow (H946) Cancer (KB) Cancer (Hep-2) Cancer (HeLa) Monkey (primary culture) Kidney (unspecified) Macaca rhesus kidney M. cynomolgus kidney M. cynomolgus leukocyte M. fuscata kidney M. cyclopis kidney Erythrocebus patas kidney E. patas leukocyte Cercopithecus aethiops kidney Baboon kidney 9, 20, 30, 33, 37, 47, 111, 112, 113, 114, 116, 147 9, 32, 33, 82, 111, 112, 113, 114, 116, 146, 147, , , , , 85, 126, 129, 132, , , 133, , , 70 12, 13, 99, 101, 105 1, 2, 12, 32, 33, 61, 62, 116, 121, 126, 129, 133, , 116, 147, 149 8, 9, 30, 33, 111, 112 8, 9, 111, , 85 73, 85 13, , 9 TABLE 1-Continued BACTERIOL. REV. Cell Suscep- Reference tibility Monkey (stable line) M. rhesus kidney (MS) C. aethiops kidney (Vero) Dog Kidney (primary) Kidney (stable DKC) Leukocyte (primary) Mouse Kidney (primary) Fibroblast (stable L) Leukocyte (primary) Hamster Kidney (primary) Kidney (stable HKC) Guinea pig Kidney (primary) Spleen (stable) Leukocyte (primary) Rabbit Kidney (primary) Kidney (Stable) Leukocyte (primary) Cattle Amnion (primary) Kidney (primary) Kidney (stable) Chicken Embryo (primary) Amnion (primary) Chorioallantois (primary) Embronic lung (primary) Leukocyte (primary) Matumoto et al. (unpublished) Matumoto et al. (unpublished) 38, 88, , , , 111, 114, , , , 32 30, Continuous Cell Lines of Human Origin Dekking and McCarthy (24) reported in 1956 the adaptation of the Edmonston strain of measles virus to the KB cancer cell line. At the same time, Black et al. (12) and Adams (1) reported passage of the virus in HEp-2 and HeLa cell cultures, respectively. Following these publications,

4 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 155 a number of reports have appeared on successful adaptation of measles virus to various cell lines of human origin, as listed in Table 1. In these systems, several passages may be required for the virus to adapt, and large inocula may be needed in the initial passages; in the early passages, the cytopathic changes are limited. Once adapted, however, multiplication of the virus generally occurs more rapidly with earlier appearance of cytopathic changes and higher yields of virus. In general, serial passage of the virus in these systems is readily accomplished when fluids of infected cultures are used. But, with the mouseadapted virus of Imagawa and Adams (59), serial transfer in HeLa cultures was difficult when supernatant fluids were employed, whereas cytopathogenesis was readily induced when infected cells were used as inoculum. The mouse-adapted measles virus of Matumoto et al. (77), however, was readily transferred serially by inoculating supernatant fluids when returned to FL cell cultures. It should be emphasized that these continuous cell lines provide particularly useful systems for the study of measles virus, because of their freedom from latent cytopathic agents, the high yields of virus that may be rapidly obtained, and the relative ease in maintenance and handling of the cells in the laboratory. In addition, continuous cell lines have an advantage over primary cell cultures, since they comprise cells of a single type. Hence, these culture systems are particularly useful in analytical studies of virus multiplication. Of interest in this connection is the observation by Rapp (100), who suggested heterogeneity of the cell population of the HEp-2 line in susceptibility to measles virus; Rapp isolated clonal strains of HEp-2 cells which differ in their response to infection with measles virus as well as in morphology and nutritional requirements. Monkey Cell Cultures In the search for an ideal system for measles virus propagation, kidney cell cultures from monkeys of different species were tried. As listed in Table 1, various species have been found to be usable. Difficulties arise again from the fact that monkey cell cultures occasionally carry spontaneous monkey agents, even measles virus. Black et al. (13) encountered these monkey agents much less frequently in African monkeys, Erythrocebus patas, and Cercopithecus aethiops tantalus, than in the macaques. Berg and Rosenthal (11) reported multiplication of measles virus in suspensions of blood leukocytes obtained from patas monkey. The MS cell line of rhesus kidney origin and the Vero cell line derived from African green monkey kidney were found to be useful for measles virus propagation in our laboratory and elsewhere. Cell Cultures ofnonhuman and Nonsimian Origin Early attempts in the laboratory of J. F. Enders failed to propagate measles virus in chick embryo or in Maitland culture of chick embryo tissues (30). Subsequently, the Edmonston strain was selected for intensive study and was carried through 24 primary renal cell passages and through 28 passages in primary human amnion cell cultures. At this time another attempt by Milovanovic and co-workers (82) to adapt the virus to chick embryo proved successful. After it was adapted to chick embryo, Katz and his associates (67) could propagate the virus in monolayers of trypsinized chick embryo cells. Subsequent attempts by many workers to adapt the virus to a variety of cell cultures of nonhuman and nonsimian origin have resulted in successful adaptation to certain culture systems, as listed in Table 1. The early study of Wright (147) showed that primary kidney cultures derived from several species of rodents (the young hamster and mouse, and guinea pigs of varying ages, embryonic to adult) develop cytopathic changes upon infection with measles virus. Tawara (129) reported successful propagation of the virus, with cytopathic effects, in the L strain of mouse fibroblastic cells and the HKC strain of hamster kidney cells. Mascoli et al. (72) propagated the virus in a continuous cell line of guinea pig spleen origin; the virus induced syncytia with inclusions. In contrast to these observations, primary cultures of rabbit kidney failed to support measles virus propagation (17, 111, 116). However, Enders et al. (34) reported rapid development of giant cells in inoculated cultures of a strain of rabbit kidney cells developed by Westwood and co-workers; further studies are desirable. Berg and Rosenthal (11) obtained no evidence for virus multiplication in suspensions of mouse, guinea pig, and rabbit leukocytes. High susceptibility of primary cultures of dog kidney was first shown by Frankel et al. (38); and this culture was employed for virus growth by Tawara et al. (128) and Norrby (88). Tawara (129) and Sato (121) reported virus proliferation with cytopathic effects in the DKC continuous line of dog kidney origin. Canine leukocytes failed to support virus growth (11). Warren and Cutchins (140) failed to propagate

5 156 MATUMOTO measles virus in primary cultures of kidney and other tissues of bovine embryos. Enders et al. (32) also reported unsuccessful propagation of the virus in bovine amniotic cell culture. However, Schwartz and Zirbel (124) succeeded in propagating the Edmonston strain in bovine kidney cell culture. Matumoto et al. (75) independently obtained similar results with the Edmonston strain and other strains of measles virus; the virus induced cytopathic effects typical of measles, characterized by multinucleated giant cells and eosinophilic cytoplasmic and nuclear inclusions. Among the culture systems available for measles virus propagation, most significant from the practical standpoint are those of chick embryo (35), as well as canine (80) and bovine renal cells (76), since viruses cultivated in these systems have lately been employed as prophylactic agents in Japan, USSR, USA, and other countries as well. CYTOPATHIC EFFECTS The cytopathic changes induced by measles virus are characterized by formation of cytoplasmic and intranuclear inclusions, formation of syncytia or multinucleated giant cells, and strand formation, consisting of spindle-shaped cells with long and irregular cytoplasmic processes and of rounded cells with granular cytoplasm. General Observations When Enders and Peebles (30) first isolated the virus, they recognized characteristic cytopathic changes in infected cultures of human and simian renal cells, primarily consisting of syncytial giant cells. Within the sheet of renal epithelial cells appeared discrete areas of varying size and shape and of nonrefractile glassy appearance, in which the cell boundaries were obliterated. The affected areas often contained numerous large and small vacuoles. After further cultivation, the affected areas were slowly extended or enlarged by coalescence with neighboring plaques, while others developed elsewhere. Degenerative changes gradually appeared within the affected areas, and finally most of the epithelial cells disintegrated. In fixed and stained preparations, the glassy areas were clearly revealed as collections of nuclei in a common cytoplasmic matrix. As many as 40 to 100 nuclei were counted in such syncytial formations. Eosinophilic inclusions were shown in the nucleus and cytoplasm. In Enders' laboratory, measles virus passaged 24 times in human renal cell cultures was adapted to human amnion cell cultures (32, 82). As passage in this cell culture progressed, in addition to the syncytial formation, increasing numbers of fusiform or spindlelike cells were noted. Spindlelike cells were more refractile, and margins of the BACTERIOL. REv. elongated cytoplasm's processes distinguishing the affected cells were somewhat irregular or shaggy and occasionally exhibited a fine beading. Usually, these changes were first seen in a few cells lying adjacent to each other. Later, much of the cell population became involved, and the process slowly terminated in complete cellular disintegration. In successive passages, spindle-cell formation tended to predominate over formation of syncytia. Nuclear inclusions were revealed in certain of the spindle cells. Similar findings in human amnion cultures were also reported by Seligman and Rapp (125). This change in cytopathogenesis on passage seems to be of rather general occurrence, since Ruckle-Enders (116) stated that measles strains of human and simian origin showed typical syncytial formation in the early passages in human or simian cell cultures, and, after 8 to 28 passages in primary cultures or continuous cell lines, they exhibited an alteration of their original cytopathic effect, with spindlecell degeneration. Frankel and West (39) showed that the predominant change observed in a stable line of human amnion cells was necrosis, which became apparent 2 to 3 days after infection with measles virus, either from tissue culture fluids or from clinical materials. Within 7 to 14 days, necrosis was virtually complete, even when dilute inocula were employed. This finding was confirmed by Mutai (85); necrosis first appeared in infected FL cell cultures as small masses packed with rounded granular cells as early as 24 hr after inoculation with the virus passaged in this culture. Significant variation has been reported in different cell systems with different strains of measles virus regarding the predominance of one or the other of these types of cytopathogenesis. As cited above, Mutai (85) noticed necrosis as the predominant cytological change in FL cells, whereas syncytial masses were far less numerous than necrotic areas and spindle cells were also found, particularly at the periphery of the cell sheet. In contrast to FL cells, the human intestinal cell line of Henle and Deinhardt demonstrated these changes, but syncytial formation was predominant. In the human conjunctival and hepatic cell strains of Chang and the H946 strain of human bone marrow cells, the predominant change was formation of syncytia which were not as large as those observed in monkey renal cell cultures. According to Okuno et al. (94) the capacity to produce cytopathic effects in FL cells was lost when the virus was carried through allantoic cavity passages in chick embryos, whereas the amniotic passage virus retained the activity. Milovanovic and associates (82) showed that simultaneous titration of amnion-adapted

6 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 157 measles virus in human amnion and kidney cultures gave similar end points, although the cytopathic changes observed were quite different: the virus produced mostly syncytia in kidney cultures, and spindle cells in amnion cultures. In adaptation of the Edmonston strain to chick embryo cell cultures, Katz et al. (67) found that, during the first four passages in this system, the virus exhibited no recognizable cytopathogenic activity. However, beginning with the fifth passage, small giant cells with spindle-cell formation and cell rounding were observed. These changes appeared consistently in subsequent passages. Musser and Slater (84) adapted the egg-passaged Edmonston strain to dog kidney cell cultures. In the early passages, the major cytological change was syncytia with eosinophilic inclusions, predominantly in the nucleus. After about eight passages, subtle changes in the cytopathic effect became apparent. Concurrent with the appearance of a spindle-cell type of cytopathogenesis, fewer localized areas of multinucleated giant cells were observed, and at this time higher yields were obtained. Similar observations were made in HeLa cells by Oddo et al. (93), who passaged the Edmonston strain, after 30 passages in human amnion cultures, in HeLa cell cultures by transferring undiluted fluid. Giant-cell formation, the outstanding feature of the cytopathic effect in the early passages, became increasingly rare and small syncytia became prevalent. After the 50th HeLa passage, syncytia containing 10 nuclei were rarely encountered. Syncytial Formation Syncytia or multinucleated giant cells in cell cultures infected with measles virus seem to be formed by fusion of mononucleated cells rather than by atypical nuclear division (8, 65, 78). This notion was definitely confirmed by Aoyama (4), who showed the sequence of cell fusion in infected monkey kidney cell cultures by time-lapse phase-contrast photomicrography. In his culture system, syncytial formation began 40 hr after infection, whereas cytoplasmic inclusions became discernible in 24 hr and intranuclear inclusions somewhat later. Cells came in contact and fused within 30 to 60 min to form a cell containing two or three nuclei. Fusion proceeded further to form cells with 5 to 10 nuclei, which congregated in the central part of the cell. Thereafter, these small giant cells fused to finally form large giant cells measuring up to 1 mm or more in diameter and containing as many as 100 nuclei. Roizman and Schluederberg (107) showed that HEp-2 cells could be infected with measles virus at any stage of cell division, mitosis being arrested upon infection, and the cells could be readily recruited into syncytia. Fusion appears to occur not only between infected cells but also between normalappearing and infected cells (4). This is further supported by the finding that, when extracellular virus is inhibited by antibody, spread of infection from cell to cell in monolayers still continues, as revealed by immunofluorescence studies (101, 116). Toyoshima et al. (134) and Karaki (65) found giant cells in FL cell cultures as early as 8 to 10 hr after infection with heavy inocula. Toyoshima et al. (135) further demonstrated that, when FL cell monolayers inoculated with heavy doses of virus were trypsinized after various periods of incubation, small fused cells could be observed as early as 1 hr after infection, and fusion proceeded rapidly for about 3 hr. However, when unstained monolayers were examined under a microscope at this time, a decreased distinctness of the cell boundaries was the only indication of cell fusion. The virus inactivated by ultraviolet irradiation also induced cell fusion to the same extent as did active virus, and by further incubation the fused cells gradually degenerated within several days, although neither infective virus nor complementfixing antigen was produced. The higher the input multiplicity was, the more fused cells were produced. However, at least 106 TCID50 of ultravioletirradiated virus per monolayer containing 3 x 105 FL cells was necessary to induce a detectable degree of giant-cell formation. Measles antibody inhibited the phenomenon when mixed with the virus before inoculation, but not after virus adsorption. The virus inactivated by heating at 56 C for 20 min did not induce the cell fusion. Neither active nor ultraviolet-irradiated virus exerted such an effect on L cells of mouse fibroblast origin. Norrby et al. (91), in concert with Toyoshima et al. (134), observed early fusion of the cells in cultures of a human embryonic cell line, Lu106, seeded with large doses of crude active virus material and also with concentrated virus material partially inactivated by incubation for 24 hr at 37C. An observation of interest was made by Schluederberg (122), who prepared a noninfectious fraction with hemolytic activity by equilibrium sedimentation in cesium chloride of tissue culture fluids infected with measles virus and tested this fraction for giant-cell-inducing activity as described by Toyoshima et al. (135). The pooled fractions diluted in phosphate buffer were incubated with HEp-2 cells for 18 hr at 37 C. Buffer alone was added to control cultures. After incubation, the cells were treated with edathamil and placed in a hemocytometer for observation. A marked difference in the amount of clumping or agglutination was noted, and distinct fusion of

7 158 MATUMOTO cells was observed. Further studies are required to determine the density distribution of the giant cell-inducing activity and also to learn whether it correlates with the lytic activity. Norrby et al. (91) described similar observations. By the CsCl density gradient technique, they prepared a fraction of low-density hemolysin with no infectivity and tested it for cytopathic effect on cover slip cultures of Lu106 cells of human embryonic origin. Such materials induced a gradually increasing confluence of cells after 2 to 8 hr of incubation at 37 C. This could not be an effect of traces of remaining infectivity, since the time of incubation does not allow extensive virus growth, and since, furthermore, no production of infectious virus and antigen could be detected after prolonged incubation of the cultures. These findings seem to indicate the presence of a fusion-inducing factor or factors which are produced in infected cells. Since the fusion-inducing activity of the preparations from infected cultures is inhibited by measles antibody, this factor seems to be a virus-specific substance, and its synthesis may be controlled by the virus genome. It seems rather unlikely that the factor is a pre-existing host substance and is merely activated upon infection, or that the factor is synthesized upon infection under the control of the host genome. It is highly probable that the factor may be identical to, or closely associated with, measles virus hemolysin. The factor seems unlikely to be released in effective amounts from the infected cells into the medium, since the giant-cell formation in infected culture proceeds rather locally, progressively involving adjoining cells. The cell fusion appears to involve enzymatic processes, since the phenomenon can be induced at a high temperature such as 37 C, by inactivated virus or hemolytic fraction, although an adequate comparative study at 37 C and lower temperatures has not been reported. The presence of specific giant cells in histological sections of human and animal tissues is considered pathognomonic for infection with measles virus. Several types of measles giant cells have been recognized: the classical Warthin-Finkelday cell seen in lymphoid tissues, the epithelial giant cell seen in sections of bronchial mucosa and smears of bronchial and nasal secretions (29), the giant cell of reticular cell origin in spleen tissues (63), and the giant cell formed by fusion of nerve or ependymal cells recognized in sections of mouse or hamster brains infected with neurotropic variants of measles virus (77, 138). Since these giant cells found in man and animals infected with measles virus are quite similar in morphological and other properties to the giant cells induced by the virus in cell cultures, it seems BACTERIOL. REV. that the same mechanism is involved in their formation, although more definitive studies are needed. Factors Affecting Cytopaihogenesis A number of workers have observed great variation of predominant cytopathic effect in different cell culture systems with various strains of measles virus. Oddo et al. (93), as cited previously, observed that giant cells, predominant in the early passages of the Edmonston strain in HeLa cell cultures, became increasingly rare and small, and spindle-cell formation became prevalent, as the passage in HeLa cells progressed. From the 60th HeLa passage, two lines were established: 100-fold diluted passage line and undiluted passage line. The former line fully regained the ability to induce giant-cell formation. The latter consistently maintained the strand-forming property through many passages. Cytological changes typical of each line were also reproduced in KB cell cultures and primary cultures of monkey kidney cells. In the interpretation of these findings, the possibility is to be taken into account that there are at least two genetically distinct types of virus, giantcell type and spindle-cell type. If this concept is correct, the stability of the giant-cell producing capacity of virus upon diluted passage may be explained by the assumption that in this passage line the giant-cell virus becomes predominant, and this type of virus is always picked up in diluted passages. However, as to the predominance of the spindle-cell virus in the undiluted passage line, data are not available regarding isolation of this type of virus from the established undiluted passage line by limiting dilution technique. In a similar experiment by Seligman and Rapp (125), the Edmonston strain passaged in HEp-2 cells showed predominantly spindle-cell formation, although giant cells were still occasionally seen. In an infectivity titration of this virus in cultures of a HEp-2 cell clone, of 17 tubes with cytopathic effects among 24 inoculated tubes, 1 tube showed giant cell formation as the sole cytopathic change. The virus from this tube continued to produce only giant cells at end-point dilution upon transfer through 10 passages in these cells. In the study of Oddo et al. (93), interesting is the observation that a marked inhibition of giantcell formation occurred in cultures of HeLa cells inoculated with 103 TcID5o of the two viruses. McCarthy (79) suggested that, if in relation to the syncytial virus the spindle-type variant showed increased production of, and perhaps decreased sensitivity to, interferon, it might gradually overgrow and blanket even a potentially faster-growing wild type. This could explain the overgrowth

8 VOL. 30, 1966 of the spindle-cell variant at high dose rates and the reversion of syncytial virus at limiting dilution. Reissig et al. (105) have shown that external factors may play a role in determining the type of cytopathic changes induced by a given measles inoculum. HEp-2 cells infected with the HEp-2- adapted Edmonston strain showed syncytial formation in Enders' medium, whereas those maintained in Eagle's medium showed spindle-cell formation, and very few syncytia were formed. The effect could be attributed primarily to glutamine by its omission from Eagle's medium, which caused syncytia as did Enders' medium. Frankel and West (39) also observed almost complete suppression of syncytial formation by the addition of glutamine to the medium in FL cell cultures infected with measles virus. However, glutamine did not affect syncytial formation by the giant-cell line of measles virus established by Seligman and Rapp (125). This line of virus seems to represent a variant which lost the glutamine sensitivity. It would be of interest to learn how to correlate these results on cytopathic effects of the environmental factors and genetic variation of the virus with the fusion-inducing factor discussed in the preceding section. Inclusion Bodies The cytopathic effects of measles virus are further characterized by formation of inclusion bodies. Enders and Peebles (30) stated in their first report on propagation of measles virus in tissue culture that examination of stained materials revealed, within the nuclei of the giant cells, significant changes that were not visible in fresh preparations. These consisted in a redistribution of the chromatin, which ultimately assumed a marginal position where it formed a dense ring or crescent that stained intensely with the basic dye. Concomitantly, the central portion of the nucleus came to be occupied by an apparently homogeneous substance, acidophilic in character, that approximated closely to the chromatin ring. In the early preparations, the acidophilic substance was only seen in small, rounded masses, distributed here and there, amid nuclear materials that approximated the normal appearance. They also noted that irregular masses of eosinophilic material accumulated in the cytoplasm. These observations have been subsequently confirmed by Cohen et al. (22), Ruckle (111), Bech and von Magnus (8), and many others. Enders and Peebles (30) noticed later that typical acidophilic intranuclear inclusion bodies were regularly seen surrounded by a clear zone or halo, when infected cultures were fixed in Bouin's MEASLES VIRUS IN CELL CULTURES 159 fluid. This was confirmed by Black et al. (13), who showed that, when the cells were fixed at a ph of about 7.2 to 7.4 in formalin or osmium tetroxide in isotonic saline, the inclusion material stained very faintly with acid dyes and occupied all of the chromatin-free areas of the nucleus; when fixatives containing acetic acid, such as Zenker, Bouin, or Carnoy fluid, were used, shrinkage of the inclusion material occurred, and a clear chromatin-free halo could be seen around the inclusion body. Measles intranuclear inclusions are quite characteristic, and can be readily distinguished from those induced by other viruses, such as adenoviruses and viruses of the herpes group (13), but resemble those induced by certain myxoviruses, such as rinderpest, canine distemper (141), or parainfluenza viruses (21, 60, 104). Measles intranuclear inclusions are eosinophilic and Feulgennegative throughout their development (13, 106). Cytoplasmic inclusions appear as masses of varying shape, ranging in size from 1 to several microns. When fixed in solutions containing acetic acid, they are surrounded by a clear halo and exhibit numerous tiny clear pores (13). Although formation of inclusion bodies is induced by measles virus, it is not necessarily induced in infected cells. In some cells, such as the human heart line, inclusions have not been seen, and the cytopathic effect is limited to spindle-cell degeneration. This host system is as sensitive to virus infection, and supports virus multiplication to the same extent, as other systems in which inclusions are found (13). Inclusion bodies appear much sooner and in a larger proportion of the cells in primary cultures of monkey kidney or human amnion than in continuous cell lines (111). Mutai (85), working with a FL-adapted virus, reported that intranuclear inclusions were observed in a relatively small number in FL cells, but hardly at all in other continuous human cell lines; cytoplasmic inclusions were numerous in infected FL cells, but infrequent in the other cell lines. Although prominent cytoplasmic and nuclear inclusion bodies are characteristic changes induced by measles virus in many cell types, information is incomplete regarding the relationship of these sites to the accumulation of viral particles or antigens. No morphological correspondence could be ascertained between inclusion bodies and accumulation of antigen (101, 107, 134) or nucleic acid (132). Toyoshima and associates (134) examined FL cell cultures infected with measles virus at an input multiplicity of about 2 TcID5o per cell by acridine orange staining coupled with ribonuclease digestion and fluorescent-antibody staining. Inclusions, ribonucleic

9 160 MATUMOTO acid, and virus antigen appeared paranuclearly at the time when increase in active virus titer could be detected. Large cytoplasmic inclusions found at the late stages were not sites of accumulation of either virus antigen or ribonucleic acid (RNA). The earliest and most evident change in the nucleus was an enlargement of nucleoli containing much ribonucleic acid. Nuclear inclusions seemed to contain very little, if any, nucleic acid, and with very few exceptions no antigen, although antigen was detected in some nuclei as faint fluorescence. Karaki (65) demonstrated that there was some correspondence between cytoplasmic inclusions and accumulation of virus antigen, although antigen was generally more diffuse in the cytoplasm, and that cytoplasmic inclusions at certain stages of development stained green with acridine orange, but this staining property was not affected by treatment of the preparations with deoxyribonuclease or ribonuclease. Electron microscopy, as will be discussed in a later section, reveals that measles inclusions do not appear to be aggregates of virus particles. INFECTIVITY TITRATION AND PLAQUE FORMATION Since the discovery of measles-specific cytopathic effects in tissue cultures by Enders and Peebles (30), infectivity titration has become feasible in tube cell cultures, with the cytopathic changes as criterion of virus infection. Although various cell culture systems can be employed for measles virus assay, their sensitivity may vary with different strains of virus or even with viruses of different passage histories derived from the same strain. For instance, Meyer et al. (81) compared infectivity titers obtained in cultures of primary human and cercopithecus monkey kidneys, continuous lines of human heart (Girardi), human amnion (CAT, FL, AV3), monkey heart (Salk), and HEp-2 and KB cells with Enders' B level vaccine virus and earlier passage virus of the same strain; they found practically comparable titers in these systems excepting human kidney, which showed a low sensitivity for Enders' vaccine virus. Matumoto et al. (76) noticed that, during serial passage of the Sugiyama strain in primary cultures of bovine kidney, the titer determined in FL cultures decreased somewhat in relation to the titer in bovine renal cell cultures. An opportunity for more precise quantitation of measles virus was opened by the study of plaque formation by Hsiung and co-workers (56). They reported plaque formation of measles virus under an agar overlay in bottle cultures prepared from kidneys of African red grass monkeys, BACrEOL. REV. Erythrocebus patas. Plaques were tiny, but distinct, with sharp boundaries, and they were obtained as early as 5 to 6 days after inoculation. After the size of a plaque increased to 2 to 3 mm in diameter, it became very distinct. The number of plaques increased up to 6 to 10 days. Plaques were also obtained in bottle cultures of rhesus kidneys, but only if the cell sheets were in excellent condition. Even so, the plaques were not as clear, and the boundaries were not as distinct as those shown on E. patas monolayers. In simultaneous tests of cells cultured under identical conditions, plaque counts in rhesus cultures were about 10% of those in E. patas cultures. This study was soon followed by that of Black (14), in which plaques were obtained with the Edmonston strain in kidney cell cultures of Cercopithecus aethiops according to the method of Hsiung et al. (56). Underwood (137) reported a plaque assay for measles virus by use of HeLa cell monolayers and chicken plasma overlay. This method, on the average, was 3.7 times more sensitive than the roller tube assay. A minimal time of 1 hr was needed for adsorption of virus on HeLa cells. De Maeyer (25) was successful in producing plaques on HeLa and WS (a continuous line of human amnion cells) cell monolayers with the Edmonston strain which had undergone passages in human kidney and amnion cultures. After 2 hr of adsorption at 37 C, the cultures were incubated under an agar overlay. On the 5th day, a second agar overlay was made. With the virus adapted to human amnion, plaques were large enough to be counted on the 7th day and increased in number until the 10th day, but not thereafter. The plaque number was proportional to the virus input. The plaque size varied slightly but did not exceed 2 mm in diameter. Plaques were also produced on monolayers of primary human amnion; they were larger in primary cultures than in the continuous cell lines. The chickadapted virus produced plaques on monolayers of chick embryo cells. Karaki (64) showed that the FL cell-adapted Sugiyama strain of measles virus produced distinct small plaques on HEp-2, FL, HeLa, and KB cell monolayers. Plaques appeared after 5 days of incubation and increased in number during the subsequent 5 days. The plating efficiency was higher with HEp-2 and KB cells than with FL and HeLa cells. In our experiences with FL and other continuous human cell lines, plaques vary considerably in size and shape, ranging from circular, distinct plaques to very tiny red spots. A virus clone, which produces relatively homogeneous clear plaques, has been established by repeated plaque cloning of the Sugiyama strain. In relation to the tiny red plaques mentioned above, the observation by Kohn and Yassky (70) is of interest that, during the early period of syncytial formation in

10 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 161 infected KB cell cultures, the uptake of neutral red added to the cultures was augmented, being concentrated in the central portion of the syncytia. The plaque-forming property may be used as a marker to distinguish the virus of live measles vaccine and virulent measles strains. Buynak et al. (19) showed that elongated plaques produced by the attenuated Edmonston virus on grivet monkey kidney cell monolayers are distinctive and quite different from those produced by the virulent Philadelphia 26 strain (small, discrete, irregularly circular plaques). Furthermore, the attenuated Edmonston strain produced small, discrete, circular plaques on chick embryo monolayers, but the Philadelphia 26 strain did not produce plaques. Rapp et al. (99) reported an infectivity assay by means of microscopic counts of immunofluorescent foci in infected HEp-2 cell monolayers. Cell monolayers prepared on cover slips are inoculated with virus and incubated under a methyl cellulose overlay. The overlay prevents secondary spread of virus, and is removed before the infected cell sheets are treated with measles antiserum and fluorescein-labeled antiglobulin for detection of virus antigen. The number of fluorescent foci is approximately proportional to the amount of inoculated infected material. Toyoshima et al. (134) and Kohn and Yassky (70) assayed infective centers by inoculating infected cells, dispersed by ethylenediaminetetraacetate, into tube cell cultures. Plaque technique, if feasible, seems to provide a more precise quantitation of infective centers. Fluorescent-antibody staining may also be employed for this purpose. PRODUCTION OF AIGENS IN INFEcTE CELL CULTURE Specific complement-fixing antigen was first demonstrated in tissue cultures infected with measles virus by Enders and Peebles (30). This finding has been confirmed by subsequent workers Ṗeries and Chany (95) described hemagglutination reaction of measles virus. Infective materials agglutinate erythrocytes from various species of monkeys, such as baboon (95, 109), rhesus (27, 109, 110), cynomolgus (109), patas (95, 109), and African green monkey (109), but not erythrocytes of sheep, chicken, guinea pig, rabbit, horse, hamster, mouse, rat, cat, cattle, and man (109). The reaction was found to be speifically inhibited by measles antiserum (27, 95, 109, 110). Measles hemagglutinin has been demonstrated in various cell culture systems, but no hemagglutinin was shown in chick fibroblast cultures, even though infective titers were as high as in the other systems which produced hemagglutinin (95, 109). Hemadsorption was also noted when monkey erythrocytes were added to cell cultures infected with measles virus (69, 95, 109). Peries and Chany (95) also noticed partial hemolysis of monkey erythrocytes agglutinated by measles virus after prolonged incubation at 37 C. Specific inhibition of this reaction by measles antibody was demonstrated. Some general properties of the hemolysin and the kinetics of its action have been studied (28, 92, 120). Immunofluorescence technique has been employed to detect measles antigens in infected cells. Cohen et al. (22) studied the development of antigens in monkey kidney cells infected with measles virus by this technique. On the whole, antigen was detected earlier by indirect fluorescent-antibody staining than by complement-fixation tests. Specific fluorescence was noted in the nuclei as well as in the cytoplasm. Enders (31) also found that immunofluorescence could serve this purpose. Rapp et al. (101) demonstrated virus antigen in either nucleus or cytoplasm, or both, of infected HEp-2 and primary human amnion cells. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Rapp and associates (101) suggested that intranuclear virus antigen was possibly associated with the nucleoli. However, Roizman and Schluederberg (107) reported that in the nucleus of HEp-2 cells virus antigen appeared as dispersed small spherical granules distinct from nucleoli. Toyoshima et al. (134) could show virus antigen in only a very few nuclear inclusions, although faintly fluorescent antigen was detectable in some nuclei. In contrast Nagahama and associates (86) could not find virus antigen in the nucleus of infected FL cells. Antigenic components produced in infected cells have been studied by fractionating infected materials. Peri6s and Chany (96) demonstrated a small noninfectious hemagglutinating unit in infected tissue cultures by ultrafiltration and ultracentrifugation. Schluederberg (122) and Schluederberg and Roizman (123) separated three fractions of complement-fixing particles, differing in infectivity and density, from concentrated cell culture materials infected with measles virus by equilibrium sedimentation in cesium chloride. The apparent buoyant density of infectious particles was approximately 1.29 g/ml, and of the two noninfectious complement-fixing antigens, 1.24 and < 1.14 g/ml. Norrby (88) showed that measles hemagglutinin represents a heterogeneous population of particles; by rate zonal centrifugation in sucrose density gradients, two fractions of hemagglutinin could be separated, called large and small hemagglutinin, respectively, with reference to their rates

11 162 MATUMOTO of sedimentation in the gradient. The large hemagglutinin carried all of the infectivity and almost all of the hemolytic and complement-fixing activities as well, whereas the small hemagglutinin exhibited mainly hemagglutinin activity. A conversion of large into small hemagglutinin was obtained by treatment with ether or ether and Tween 80 (88, 144). Kitawaki et al. (68) obtained highly purified preparations of small hemagglutinin produced by ether-tween 80 treatment. The artificially produced small hemagglutinin differed in some respects from the naturally occurring or native small hemagglutinin (68, 88). Using equilibrium centrifugation in CsCl gradients of the concentrated tissue culture preparations of measles virus, Funahashi and Kitawaki (42) showed that hemagglutinin is located in two main fractions with densities of 1.19 and 1.24 g/ml. In a similar study, Norrby (90) demonstrated two visible bands; one was sharp, located at a density of 1.30 g/ml and exhibited mainly hemagglutinating activity, and the other band was broad and extended from a density of 1.25 all the way down to 1.13 g/ml. Infectivity was present as a sharp peak in the high density part of this band, and hemolyzing and complement-fixing activities showed maxima at 1.23 to 1.24 g/ml. The latter two activities, however, were present throughout the broad band, although there were some differences with regard to their distribution. Tween 80 and ether treatment of the virus preparation caused a complete disappearance of the broad band, both visibly and in activity assays. Only the sharp band with a density of 1.29 to 1.30 g/ml remained, and it exhibited a markedly increased hemagglutinating activity and, in addition, complement-fixing activity. One more band was found which presumably constitutes soluble antigen. In their further study, Norrby et al. (91) showed that the large hemagglutinin fraction appeared rather homogeneous, biological activities being concentrated in fractions with densities of 1.20 to 1.25 g/ml. This figure of density is significantly different from 1.29 g/ml for the infectious particle fraction obtained by Schluederberg and Roizman (123), but the reason for this discrepancy is obscure. In the same range of densities, an accumulation of RNA was revealed, whereas no significant amounts of deoxyribonucleic acid (DNA) could be detected. RNA in this fraction could be eliminated by adsorption with monkey erythrocytes, whereas, if the material was first treated with Tween 80 and ether, no change in RNA content was demonstrated after erythrocyte adsorption. These findings suggest that the nucleic acid of measles virus is RNA. BACrERIOL. REV. ELECTRON MICROSCOPY OF INFECTED CELLS The most outstanding feature of cytological changes in cells infected with measles virus as revealed by electron microscopy is the appearance of filamentous structures in nuclear and cytoplasmic inclusion bodies. Reissig (106) briefly reported that nuclear inclusions were found not to be aggregates of viruslike particles, but to correspond to areas of low electron density, where a loss of the normal chromatin network of the cell nucleus has occurred. Kallman et al. (62) showed that the fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated HeLa cells infected with measles virus consisted mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusion, sometimes of a two-dimensional, nearly orthogonal arrangement. These observations have been confirmed by subsequent workers in infected human amnion cells (6, 116), HeLa cells (116, 128), KB cells (87), and dog kidney cells (130, 131). Depending on the plane of sectioning, a particulate-like appearance in ordered array or crystal-like group was seen in the inclusion bodies (6, 116, 128, 130). These filamentous structures were shown to have a diameter of 150 to 200 A and a central hole 100 to 150 A in diameter (130, 131). The nature of these structures seen in the inclusions is obscure, but obviously the strands cannot be fully formed virus because of their size and morphology. Baker et al. (6) showed particles on, or outside of, the cell wall that might be interpreted as mature virus because of their morphology and size. They exhibit double membranes, separated by 50 A, an internal granular structure, and have an approximate diameter of 1,200 A. Ruckle- Enders (116) also observed viruslike particles approximately 0.1 A in diameter near the cell surface or outside of human amnion cells. These observations are suggestive of viral maturation at the cell wall, a phenomenon characteristic of the myxovirus group, in which measles virus is classified. The size of these particles seen by Baker et al. (6) and Ruckle-Enders (116) is comparable with the figure of 1,400 A determined by filtration (10) and by ultracentrifugation (83). The fine structure of the measles virus particle was described by Waterson (143), who studied virus preparations, negatively stained with phosphotungstate, by use of an electron microscope. The virus particles are approximately circular or oval, but they have no regular form (Fig. 1). Their overall diameter is 1,200 to 2,500 A. A well-de-

12 s::ik- } S! < VOL..i 3 s I... *.., :.^. S. *:l $.$... i_11 i'. :' :': _11 -_1E :E _ 30,1966 j=.6:' s Xk < *. 2.9i:-i,.,g.-. je i:. _ ig!,. 3S,;kd - _i1 _-... _1sswNi,?R.... :-S MEASLES,'- de ;.,3.,veA.:-P.. i....,b7s;;o.- x... i i:-. - l1 _*. j le i VIRUS _ i' ;g l.s.^a _g, _E. _=X11 oin l -gi' kj. CELL 2_..: x.c ->B$d; :e CULTURES.: _.^, ik%. l_'. : ':... dc:, k7s5 l/_rls' dsswlers_.: g ' A -: ;...::::w:.. zi j- : ','i!!_itt'! ----=XSe-! j.,,.,. ;, & *... fd: ha... 11_- E8 8:b' ^,Bi 111!1 _ X - oo *S- ig _ il _ 11 XgYS.Ek, n -o-c; I ] C BafSU9to fi:::_.-n dg^: w.; 9!...:8.'.f *!::'^ l, ::i: :ZMi_, sm- ss...1@a 5&lC ;.&dls:_,,es ks; I11@g_ gbk u r.2. il23. If z: ser. 9t'}.a_ q''i.#_ SiB_ ii :S '.,l bx ^ _!s_m.4ir<iu k; ;1_.z., sa1_ Jb...n_ ;'':E,, gz I 3._}X/l i &..1,ise.:eZk.9.:.'...i., i emoos :.. ::.::.n'... ';k 26.,>&=2p6;! d.31 i :101 31! k2 ki z.-. FIG. 1. Measles virus particle negatively stained with phosphotungstate. X EZectron micrograph provided by Y. Hosaka, Osaka University. fined membrane, about 100 A thick, is shown at can be resolved in the intact particles. Ln some, the periphery of the particle, and numerous short however, concentric rings can be seen, each ring projections are seen from the surface of the mem- having a thickness of 120 to 150 A. From partially brane. In general, little of the internal structure disrupted particles, a rod-shaped structure is pro-

13 164 MATUMOTO jected. The same structure can be shown in virus preparations treated with ether and Tween 80 (144) (Fig. 2). This structure has a diameter of 170 to 180 A, and a central hole about 50 A in diameter. The outside of the rod shows regular serrations, with a periodicity of about 55 A. FIG. 2. Measles virus capsid. Virus preparation was treated with ether and Tween 80 and stained negatively with phosphotungstate. X 225,000. Electron micrograph provided by Y. Hosaka, Osaka University. Hosaka (53, 54) confirmed these observations and, in addition, showed that the helical capsid is contained in a sheath, being at variance with that of parainfluenza virus. These observations indicate a remarkable resemblance of the fine structure of the measles virus particle to that of myxoviruses; in particular, the structure of the internal BACTERIOL. REV. component coincides with that of large myxoviruses, such as mumps, Newcastle disease, and parainfluenza (55, 144). The viruses of rinderpest (97, 145) and distemper (23, 89, 145), which have been found to be closely related to measles virus, have the same fine structure of their virion as measles virus. QUANTITATIVE ANALYSIS OF Vmus GROWTH Growth Curve Measles virus in cell cultures grows at a lower rate than most other viruses. For instance, Black (14) found the first active virus after 15 to 18 hr of incubation at 37 C in HEp-2 cell cultures infected at an input multiplicity of 1.5 TCID5o per cell. Even then, infectious virus could not be detected in the fluid phase, but only in the cells. Virus was first found in the fluid phase of the culture after 27 to 30 hr of incubation. Virus titers of the fluid and the cells increased until the 48th hr. Thereafter, both titers remained steady for several days. The cell-associated virus always titered higher than the extracellular virus. Cytopathic changes were not discernible until after peak titers had been reached, but extensive degeneration was noted before the titers started to decline. Similar observations in HEp-2 cells were also described by Black et al. (13). In an experiment with FL cells infected with a FL-adapted strain of measles virus at an input multiplicity of about 2 TCID5o per cell, Toyoshima et al. (133) first noted a rise in titer at 20 hr, and the maximal titer was reached in 2 to 4 days. The titer of the fluid was always much lower than that of the cells. Complement-fixing antigen was first shown at 24 hr and reached a maximal titer in 2 days. Cytopathic changes were first detected at 24 hr, and cytoplasmic and nuclear inclusions at 24 and 48 hr, respectively. Under similar conditions, production of active virus and complementfixing antigen and appearance of cytopathic changes were somewhat slower in HeLa cells than in FL cells. In another experiment, after massive infection of FL cells at an input multiplicity of about 60 TCID5O per cell, the first infectivity rise was shown as early as 12 hr after inoculation; the peak titer, at 30 hr (134). Karaki (65) obtained similar results with FL cells infected with the Sugiyama strain at an input multiplicity of 6 plaque-forming units (PFU) per cell. Kohn and Yassky (70) mixed 2 x 105 TCm5o of virus and 1.8 x 106 KB cells in suspension and plated the mixture in petri dishes after 1 hr of adsorption. Rise in infectivity titer occurred between 10 and 17 hr in both fluid and cells. Cell-associated virus increased to a maximum at 48 hr, when practically all cells in the culture registered as infective cen-

14 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 165 ters. Virus in the fluid increased much more slowly for the next 3 days. Similarly, Rapp and Gordon (98) reported the first appearance of active virus in the cells at 12 to 18 hr after infection of HEp-2 cells and in the fluid after 30 hr. Figure 3 illustrates the representative results of our experiments in FL cell cultures infected at various input multiplicities with the Sugiyama strain of measles virus which had been adapted to these cells. The figure demonstrates the features of the growth curve typical of adapted measles virus in cultures of continuous cell lines, which cycle of virus multiplication is completed within 24 hr. Latent Period Our present knowledge is very limited regarding the processes occurring in the period between inoculation of virus and production of initial active virus. By means of plaque assay under a variety of conditions, it has been shown that measles virus has an attachment time of 1 to 5 hr (25, 64, 137). In our study with FL cell monolayers and the Sugiyama strain fully adapted to those 7 Cell-assc 6 Exp. 1 5 / 4 E Ext 3 I Hours after infection FIG. 3. Growth ofmeasles virus (Sugiyama strain) in FL cell cultures. have been described by the previous workers, as cited above. The infectivity titer of cell-associated virus in the culture increases more rapidly, is always much higher, and reaches a plateau earlier, than that in the fluid phase. The infective titers in both cells and fluid show increasingly earlier rise and greater rates of increase, and reach plateaus earlier, as the input multiplicity increases. The curves of experiment 2 in Fig. 3 were obtained with a high input multiplicity of 38 PFU per cell and are considered to represent the one-step growth, since 100% of the cells were shown to be infected by fluorescent-antibody staining. The latent period is estimated to be about 10 hr, and a cells, an attachment time of about 1.5 hr was obtained. Toyoshima et al. (134) determined the number of infected cells by infective center assay in tube cultures; about 10% and almost 100%, respectively, of FL cells in monolayers were registered as infected cells, when assayed 6 hr after inoculation, with input multiplicities of about 0.6 and 6 TCID5o per cell or greater. According to Underwood (137), more rapid multiplication of the virus is obtained by mixing virus and cells before plating than with later inoculation after the cell sheet is completed. A similar observation was obtained with hog cholera

15 166 MATUMOTO virus (74), but the reason for this finding is obscure. Those very limited data in the literature indicate that, first of all, detailed studies of the kinetics of virus adsorption, reversible and irreversible, onto the cells are urgently needed in order to understand the initial processes of infection with measles virus. The mechanism of penetration into the cells and uncoating of the virus awaits clarification. The fact that actinomycin D, an inhibitor of DNA-dependent RNA synthesis, does not inhibit the replication of measles virus (3, 77a) suggests that de novo synthesis of hostcoded substances, e.g., uncoating enzyme as suggested in the case of vaccinia virus, is not involved in uncoating of measles virus. By analogy with influenza and other viruses of the myxovirus group, the mechanism of measles hemagglutination might be worthy of investigation as a model of the initial processes in measles infection of susceptible cells, although agglutinability by measles virus of monkey erythrocytes is not affected by neuraminidase, which can destroy the receptor for influenza viruses. Similarly, investigation of measles hemolysin might also be rewarding for elucidation of early processes in measles infection. Intracellular Multiplication of Virus In one-step growth experiments in certain host cell-virus systems, cell-associated virus increases for about 10 hr after a latent period of about 10 hr, as shown in Fig. 3. However, the most peculiar feature noticed here is that the peak titer attained is only a few infectious units per infected cell (70, 77a, 134). To estimate the per cell yield, virus released from the cells into the medium should be taken into account. Although its estimation is complicated because of the relative lability of measles virus, theoretically the per cell yield of active virus could be estimated under certain assumptions regarding the production rate of intracellular active virus and the release rate of active virus accumulated intracellularly, and by making allowance for thermal degradation of virus in the fluid phase. But this type of study is not available yet. In one-step growth experiments, such as that illustrated in Fig. 3, the amount of active virus in the fluid phase is exceedingly small in relation to the amount of intracellular active virus over the period of active multiplication of virus, and the loss of infectivity by thermal degradation in the fluid phase may be at most 90% of released active virus, as will be discussed later. Hence, the contribution of released virus may be virtually negligi- BACTERIOL. REV. ble in estimating the per cell yield, and hence the per cell yield appears actually to be only several infective units. This does not necessarily mean that the average number of active virus particles produced per cell is small, since there is the possibility that the efficiency of the currently used methods for infectivity titration is low. However, at present this is not known. Kohn and Yassky (70) reasoned that one infective unit would be equivalent to about 10 to 200 particles of measles virus, because, according to Rosanoff (109), one hemagglutinating unit of measles virus is equivalent to 104 '3 to infective units and, for about 107 erythrocytes per tube, as used by Rosanoff, the minimal number of virus particles necessary to produce visible hemagglutination would be of the order of 106. Even so, noninfectious but still hemagglutinating particles would naturally be contained in the virus preparations, and hence the number of infectious particles per infective unit cannot be known. Therefore, the notion that the per cell yield is actually very small still remains, and several other possibilities will be considered. The first to be considered is the possibility that the nutritional conditions of the host cell-virus systems employed in these studies might be far from optimal for multiplication of measles virus. Warren (142) stated that a healthy, luxuriant cell sheet, richly fed with serum protein, is essential for obtaining maximal yields of measles virus. The virus yields from HEp-2 cell cultures have been reported to be at least 10-fold higher in Eagle's medium with 5 or 10% calf serum than in Enders' medium composed of 35% bovine amniotic fluid, 5% beef embryo extract, 25% heated horse serum, and 35% Hanks solution (105). These results indicate desirability of further investigations of the nutritional requirements for measles virus propagation. In this connection, an observation of interest has been reported by Sabina et al. (119); in HEp-2 cell cultures, amino acid amidase activities were found to be sensitive to varying degrees to the action of measles virus. Leucinamidase showed an activity reaching a maximum about 16 hr after infection, and then decreasing rapidly. The peak activity of glutaminase was found at 24 hr after infection, with a subsequent decline in activity. Moreover, after inoculation of virus, no increase in glycinamidase activity was detected. Similarly, maximal activity was observed earlier with malic dehydrogenase than with amino acid amidase. The incubation temperature is another factor to be considered, since Underwood (137) showed

16 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 167 that somewhat higher infectivity titers were obtained when infected cultures were maintained at 32 to 33.5 C rather than at 37 C. The heterogeneity of the cell population in susceptibility to measles virus infection may be one of the factors to be considered. Rapp (100) has isolated several clonal strains of HEp-2 cells which differ in their response to infection with measles virus. One line of cells produced more than 10,000 times as much virus as some other lines. The amount of virus necessary for initiating infection of the various clones differed by as much as 1,000-fold, and insusceptibility was correlated with a delayed appearance of cytopathic effect. The cytopathic response also differed qualitatively among clonal types. It was found possible to synchronize cell division of the clone exhibiting the shortest generation time by cultivating cells after maintenance at a low temperature. The clone that has been synchronized is also the most sensitive to infection with measles virus. There appears to be a correlation between generation time, time required for cytopathic response, and amount of measles virus produced 7 days after infection. These findings suggest that the cell cultures employed in the growth experiments with measles virus, cited above, conceivably consisted of cells with varied susceptibility to infection with measles virus, and relatively resistant cells contributed to decreasing the virus yield per cell in these host-virus systems. It is obviously desirable to reinvestigate the growth of virus with the use of a highly sensitive cell clone. The possibility may be considered that some process, such as interferon production, proceeds along with virus multiplication in infected cultures and exerts an inhibitory effect on virus growth. De Maeyer et al. (26) showed production of interferon in cell cultures infected with measles virus. A more detailed discussion of interferon produced by measles virus will be found in the next section. Release of Intracellularly Produced Virus Growth curves of measles virus in cell cultures show that the infectivity titer of the fluid phase is much lower than that of the cells, and its increase is much slower than that of cell-associated virus. On the one hand, active virus accumulates in the fluid by release of intracellularly produced virus from the cells and, on the other hand, the released virus loses infectivity at a certain rate by thermal degradation. Thermolability of measles virus may conceivably affect the curve of fluid-phase infectivity. Ruckle and Rogers (112) reported a loss of about 90% of infectivity of measles virus per day at 37 C; under similar conditions, Musser and Underwood (83) observed 90% loss in 13 hr, and Kohn and Yassky (70), 90% in 13 hours. Black (14) reported 50% loss in about 2 hr. However, there still is reason to believe that the rate of virus release from the cells is exceedingly low. In one-step growth, as illustrated in Fig. 3, the amount of thermally degraded virus found in the fluid at a certain time may be less, probably much less, than 90% of virus released during the period of at least about 30 hr after infection. Even if an allowance of 90% inactivation is made, the amount of released virus is considered to be much lower than that of intracellular virus, suggesting an exceedingly low rate of virus release, and hence the majority of intracellularly produced virus would remain cell-associated until finally released by disintegration of the cells. Release of active virus from an infected cell seems to be a continuous process, lasting a certain period of time. When the medium is removed from infected cultures at various time intervals and the cultures are washed and reincubated for 1 hr with fresh medium, the virus titer in the singlehour harvests is close to that in the fluids representing long periods of harvest (13). Similar observations have also been reported by other workers (142). From this type of experiment, a reasonable estimate of the rate of release can be obtained. It is desirable to carry out experiments under conditions enabling us to obtain a one-step, or nearly so, growth curve. EFFECTS OF ACTINOMYCIN D ON REPLICATION OF VIRUS Certain RNA-containing viruses, such as polio (103), mengo (40, 52, 102, 103), Newcastle disease (7, 71), parainfluenza (18), Chikungunya (41, 43, 49), and Semliki Forest viruses (41), have been shown to replicate in cell cultures treated with actinomycin D, whereas DNA-containing viruses, such as vaccinia (102, 103) and herpes simplex viruses (108), are inhibited by this antibiotic. In contrast to the viruses of Newcastle disease and parainfluenza, influenza virus is inhibited by actinomycin D (7). Anderson and Atherton (3) have shown in a short note that measles virus is able to proliferate in cells treated with actinomycin D; the HEp-2 adapted Edmonston strain multiplied to a titer of PFU/ml in chick embryo fibroblast monolayers pretreated with the antibiotic, whereas normal cell cultures yielded fewer than 10 PFU/ml. The enhancement of measles virus replication by actinomycin D has also been shown in our laboratory (77a). The Sugiyama strain of measles virus adapted to FL cells was inoculated into FL

17 168 MATUMOTO cell monolayer cultures. As illustrated in Fig. 4 and 5, a more rapid increase of cell-associated virus is the most outstanding feature of the growth curves in the cell cultures treated with the antibiotic, either before or after virus infection, in comparison with the replication of virus in untreated cells. The latent period appears to be the same, or not much different, in the treated and control cultures, although this is difficult to decide, since the residual infectivity obscures the initial rise in titer. Subsequent virus titers in the 7 BACTERIOL. REV. bromo-, and 5-iodo-2'-deoxyuridine, which are inhibitory not to RNA viruses, but to DNA viruses (48, 71). In a chemical analysis of their fraction of measles virus particles prepared by equilibrium centrifugation in CsCl gradients, Norrby et al. (91) have provided evidence that the nucleic acid of measles virus is RNA, as discussed earlier. Actinomycin D has been shown to inhibit DNA-dependent RNA synthesis (45, 58, 103). This inhibition is believed to involve the forma- 6 5 Cell-associated -1 as Control To 4 Extraceliuh ar virus b Actinomycin D 0.06 ug/ml 3 2 F 1.8x 106 cells/bottle 8.0 x 105 PFU/bottle 0.44 PFU/cell Hours after infection FIG. 4. Effect ofactinomycin D on measles virus growth (1). FL cell monolayers, after virus adsorption, were incubated at 36 C in medium containing actinomycin D, or in medium without the antibiotic (Sugiyama strain). cells are always much higher during the period of active virus growth, and the peak titer is usually about 1 log unit higher in the treated cells than in the normal cells. The finding of Anderson and Atherton (3) and ourselves that measles virus is able to replicate in cells treated with actinomycin D suggests that measles virus is an RNA virus. This is in accord with certain other observations. Thus, the fine structure of measles virus particles resembles remarkably that of large myxoviruses, which are RNA viruses (143). The growth of measles virus in cell cultures is not inhibited by 5-fluoro-, 5- tion of a complex between DNA and the antibiotic (46), without apparently interfering with the synthesis of DNA but impairing some other function of cellular DNA, notably RNA synthesis (103). Hence, replication of measles virus, as of certain other RNA viruses, is considered to be independent of DNA-dependent RNA synthesis of the host cell. Another feature of interest noted in the replication of virus in cells treated with actinomycin D is that the treated cells not only support virus replication but also enhance it. As will be discussed in the following section, interferon is pro-

18 VOL. 30, 1966 duced in cultures of cells, such as primary chick embryo cells, human amnion cells, and continuous cell lines (HeLa, WS), infected with measles virus (3, 26). Because actinomycin D inhibits cellular RNA synthesis and hence protein synthesis, it would be expected to inhibit production of interferon, and it has been shown by Anderson and Atherton (3) that this is the case in the measles virus-chick embryo cell system, as was observed in chick embryo cell cultures with Chikun- MEASLES VIRUS IN CELL CULTURES 169 feron is produced in cells infected with measles virus. Ho and Enders (50) have briefly mentioned that fluids of tissue culture infected with measles virus suppress growth of poliovirus and its cytopathic effect. Subsequently, De Maeyer and Enders (26) were able to confirm these findings and to demonstrate that they depend upon the presence of interferon. Interferon was demonstrated in primary cultures of human amnion cells and in cultures of HeLa and WS cells (continuous 7 6 Actinomycin D 0.1 pg/ml 360C, 4 hrs, before infection Cell-associated virus 5 - L.9 t-l 0r 4 3 (Cont ro) Extraceliular virus x 106 cells/bottle 2.0 x 106 PFU/bottle 1.4 PFU/ceil Hours after infection FIG. 5. Effect ofactinomycin D on measles virus growth (2). FL cell monolayers were treated with actinomycin D at 36 C for 4 hr, rinsed twice, and incubated at 36 C after inoculation with measles virus (Sugiyama strain). Cell cultures not treated with the antibiotic served as controls. gunya (41, 49), Semliki Forest (41), and Newcastle disease viruses (41). However, no data are available at present concerning the susceptibility of measles virus to interferon activity and the effect of actinomycin D on the activity of interferon in these virus-host systems. INTERFERON IN REPLICATION OF VIRUS Many virus-cell systems have been shown to elaborate interferon, a substance which interferes with the replication of many viruses in related cell systems (51). It has been reported that interline of human amnion cells) infected with measles virus. This inhibitor has been separated from infectious particles and complement-fixing antigens produced in the same cultures. It is not neutralized by measles antiserum. Trypsin destroys interferon activity, but ribonuclease and deoxyribonuclease do not. Heating for 2 hr at 56 C has no effect on interferon titer, in contrast to that of measles virus infectivity which is rapidly destroyed under these conditions. It does not inactivate the test viruses, such as poliovirus and Sindbis virus, when mixed with it, but reduces

19 170 MATUMOTO BACTERIOL. REv. the extent of cytopathic changes as well as quantity of the virus produced. Anderson and Atherton (3) have shown production of interferon in chick embryo fibroblast monolayers infected with the Edmonston strain of measles virus adapted to HEp-2 cells. They have also demonstrated that treatment of chick fibroblasts with actinomycin D prior to inoculation with measles virus results in inhibition of interferon production, accompanied by enhancement of viral growth, as previously reported with Chikungunya virus (49). Thus, actinomycin D- treated cells yielded PFU/ml of virus with undetectable interferon production, whereas normal cell cultures produced fewer than 10 PFU/ ml with an interferon titer of 1:12. A more detailed discussion of the action of actinomycin D is found in the preceding section. These findings suggest that monolayers of chick embryo fibroblast showing a high degree of resistance to measles virus are rendered more susceptible by suppression of interferon production. The very low yield of active measles virus in certain continuous cell lines, as discussed in the preceding section, might be explained, in part, by concomitant production of interferon, but further investigations are needed, particularly regarding the production of interferon upon infection with measles virus and the susceptibility of the virus to interferon activity in these host systems. It has been suggested by Enders (36) that the interferon found associated with the chick cell line of measles virus may be related to the attenuation of that line. As mentioned earlier, McCarthy (79) suggested interferon as a factor responsible, at least in part, for the overgrowth of spindle-cell variant upon passage in HeLa cells at high dose rates and for the reversion of syncytial virus at limiting dilution. CARRIER STATE CULTURE A number of animal viruses can establish infection of cell cultures that result in the persistent multiplication of the virus, while the culture continues to survive and to grow (139). Rustigian (118) reported that in HeLa cell cultures infected with measles virus, after marked destruction of the cells, new cell growth began from surviving cells, and cultures could be continued indefinitely. In the original carrier culture, no active virus was found in the medium, but complement-fixing antigen, hemagglutinin, and infectious virus were regularly found when the cells were disrupted. The cultures were not cured by addition of antibody to the medium. A clone of cells was isolated from the culture after many subcultures in antibody-containing medium. This clone was found to have abundant complementfixing antigen in the cells, but no measurable hemagglutinin and no infectious virus. No infectious virus reappeared in these cells after continued subculture in antibody-free medium, and the cells did not transmit infection when they were plated on indicator cells. In this subline, 80% of the cells had antigen in their cytoplasm, demonstrable with fluorescent antibody, and 60 to 80% had cytoplasmic inclusions. These findings seem to indicate that in this subline transmission of virus is from cell to daughter cell through cell division. The virus, however, does not go through the usual cycle of multiplication and produce active virus; virus infection in this system appears to be under some sort of intracellular regulation or control. To elucidate further the cell-virus relationship in this system, it would be pertinent to study the response of these cells to homologous and heterologous viruses, and the presence or absence of interfering factors in the culture. The most fundamental question pertains to the relationship between the viral genome and the host cell genome. SUMMARY AND FUTURE OUTLOOK Much has been achieved in studies on the replication of measles virus during the past decade since consistent work with this virus became feasible after the findings of Enders and Peebles (30) in However, our present knowledge on this subject is still very limited in comparison with that of certain other viruses, such as poliovirus, vaccinia virus, or influenza virus. The mechanism of the multiplication of measles virus is presumably the same in the essential scheme as that of the viruses in the large myxovirus group, since the ultrastructure of the virus particle and certain other properties indicate that measles virus is a member of this group. The data accumulated so far, however, are grossly inadequate even to draw a rough sketch of the mechanism of measles virus growth, although the general pattern of the growth curve has been studied in certain cell culture systems. At present, the data are scanty regarding the kinetics of adsorption of the virus on susceptible cells, and little is known about the penetration and decoating of infecting virus. Hemagglutination and hemolysis of simian erythrocytes by measles virus may be useful as a model in the study of the early processes of measles infection. Actinomycin D does not inhibit the replication of measles virus, a fact suggesting that multiplication of the virus is independent of DNA-dependent RNA synthesis of the host cell. This sequence of biochemical events occurring in the intracellular synthesis of virus-specific substances,

20 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 171 such as viral RNA and proteins, awaits elucidation. Immunofluorescent technique has demonstrated virus antigen in infected cells; specific fluorescence is predominantly in the cytoplasm. Most workers found virus antigen also in the nucleus, but some did not; the reason for this discrepancy is obscure. In this connection, it is worth noting that S antigen and hemagglutinin antigen of fowl plague virus form in the nucleus and cytoplasm, respectively (16), whereas multiplication of parainfluenza, mumps, and Newcastle disease viruses, which belong to the large myxovirus group, does not include production in the nucleus of antigen detectable by fluorescent antibody (136). Antigenic structure of measles virus has been studied by complement fixation, hemagglutination inhibition, or hemolysis inhibition tests, and certain distinct antigenic components, including the virus particle itself, have been separated from infected materials. The basic antigenic units of primary importance seem to be the hemagglutinin and the protein of the capsid. However, the latter antigen in particular has not been adequately characterized as yet. To analyze the sequential development of different virus antigens in infected cells, it is prerequisite to characterize these virus antigens and to develop the techniques for their identification by staining with fluorescent antibody. The most outstanding feature of morphological changes in infected cells is formation of cytoplasmic and nuclear inclusion bodies, but information is incomplete regarding the relationship of these structures to the accumulation of viral particles, antigens, or nucleic acid. Electron microscopy has revealed that measles inclusions do not appear to be aggregates of virus particles, and instead contain filamentous structures of unknown nature. Maturation of the virus seems to occur at the cell wall, as suggested by electron microscopy. Release of virus from cells is considered to be a continuous, but exceedingly slow process, and the majority of active viruses produced in the cell seems to remain cell-associated, until finally released by disintegration of the cell. Interesting observations have been made suggesting that syncytial formation, characteristic of the cytopathic effects of measles virus, is due to a fusion-inducing factor or factors which are produced in infected cells, presumably under the control of the virus genome, and the factor seems to be closely associated with hemolytic activity of measles virus. In the light of these past experiences, it can be seen that improvement of experimental techniques is urgently needed. In analytical study of virus replication, it is necessary very often that experiments be performed under the conditions enabling us to obtain a one-step growth of virus; with measles virus, however, it is not so easy a task at present, since a high-titered infectious material necessary for such a type of experiment can be obtained only with difficulty. For quantitative analysis, well-controlled host cell-virus systems are necessary. To meet these requirements, further investigations are needed, for instance, to learn the nutritional requirements for the replication of measles virus, to obtain a relatively uniform population of host cells with high susceptibility to measles virus, and to obtain purified virus preparations, consisting predominantly of accurately and easily assayable infectious units. Proliferation of measles virus is complicated, since interferon is produced in cells infected with this virus. The low yield of measles virus generally observed in various cell cultures might be explained, in part, by concomitant production of interferon, but further investigations are needed. Measles virus can establish infection of cell cultures that results in the persistent proliferation of the virus, while the host culture continues to survive and to grow. It is of interest that a carrier culture of HeLa cells has been established in which transmission of measles virus seems to be from cell to daughter cell through cell division, and in which virus does not go through the usual cycle of multiplication and produce active virus. Studies on carrier cultures would not only provide needed information on various possible relationships of measles virus to cells, but also might be useful in the study of viral oncogenesis. AcKNowLEDGMENTs I thank Masahiko Oda of the Institute for Infectious Diseases, University of Tokyo, for criticism and assistance in preparing this review, and Yasuhiro Hosaka of the Research Institute for Microbial Diseases, University of Osaka, for providing the electron micrographs of measles virus used in this review. LrrERATURE CrrED 1. ADAMS, J. M Giant cell pneumonia. Clinicopathologic and experimental studies. Pediatrics 18: ADAMS, J. M., AND D. T. IMAGAWA Immunological relationship between measles and distemper viruses. Proc. Soc. Exptl. Biol. Med. 96: ANDERSON, C. D., AND J. G. ATHERTON Effect of actinomycin D on measles virus growth and interferon production. Nature 203: AOYAMA, Y Changes of cultured cells infected with measles virus. Japan. J. Exptl. Med. 29: ARAKAWA, S Recent advances in measles

21 172 MATUMOTO virology. Ergeb. Mikrobiol. Immunol. Exptl. Therap. 38: BAKER, R. F., I. GORDON, AND F. RAPP Electron-dense crystallites in nuclei of human amnion cells infected with measles virus. Nature 185: BARRY, R. D., D. R. IVES, AND J. G. CRUICK- SHANK Participation of deoxyribonucleic acid in the multiplication of influenza virus. Nature 194: BECH, V., AND P. VON MAGNUS Studies on measles virus in monkey kidney tissue cultures. I. Isolation of virus from 5 patients with measles. Acta Pathol. Microbiol. Scand. 42: BECH, V Studies on measles virus in monkey kidney tissue cultures. 2. Development of cytopathic changes and identification of the cultivated agents by complement fixation tests. Acta Pathol. Microbiol. Scand. 42: BENYESH, M., E. C. POLLARD, E. M. OPToN, F. L. BLACK, W. D. BELLAMY, AND J. L. MELNICK Size and structure of ECHO, poliomyelitis, and measles viruses determined by ionizing radiation and ultrafiltration. Virology 5: BERG, R. B., AND M. S. ROSENTHAL Propagation of measles virus in suspensions of human and monkey leukocytes. Proc. Soc. Exptl. Biol. Med. 106: BLACK, F. L., M. REISSIG, AND J. L. MELNICK Propagation of measles virus in a strain of human epidermoid cancer cells (Hep-2). Proc. Soc. Exptl. Biol. Med. 93: BLACK, F. L., M. REISSIG, AND J. L. MELNICK Measles virus. Advan. Virus Res. 6: BLACK, F. L Growth and stability of measles virus. Virology 7: BLATTNER, R. J Recent development in the study of measles virus. J. Pediat. 51: BEITENFELD, P. M., AND W. SCHXFER The formation of fowl plague virus antigens in infected cells, as studied with fluorescent antibodies. Virology 4: BROWN, L. V Pathogenicity for rabbit kidney cell cultures of certain agents derived from "normal" monkey kidney tissue. I. Isolation and propagation. Am. J. Hyg. 65: BUKRINSKAYA, A. G., AND V. M. ZHDANOV Shortening by actinomycin D of latent period of multiplication of Sendai virus. Nature 200: BUYNAK, F. B., H. M. PECK, A. A. CREAMER, H. GOLDNER, AND M. R. HILLEMAN Differentiation of virulent from avirulent measles strains. Am. J. Diseases Children 103: CARLSTR6M, G Comparative studies on measles and distemper viruses in suckling mice. Arch. Ges. Virusforsch. 8: BACrERIOL. REV. 21. CHURCHILL, A. E Intranuclear inclusions produced by bovine para-influenza. Nature 197: COHEN, S. M., I. GORDON, F. RAPP, J. C. MA- CAULAY, AND S. M. BUCKLEY Fluorescent antibody and complement fixation tests of agents isolated in tissue culture from measles patients. Proc. Soc. Exptl. Biol. Med. 90: CRUICKSHANK, J. G., A. P. WATERSON, A. D. KANAREK, AND D. M. BERRY The structure of canine distemper virus. Res. Vet. Sci. 3: DEKKING, F., AND K. MCCARTHY Propagation of measles virus in human carcinoma cells. Proc. Soc. Exptl. Biol. Med. 93: DE MAEYER, E Plaque formation by measles virus. Virology 11: DE MAEYER, E., AND J. F. ENDERS An interferon appearing in cell cultures infected with measles virus. Proc. Soc. Exptl. Biol. Med. 107: DEMEIO, J. L., AND T. A. GOWER Hemagglutination by measles virus. Virology 13: DEMEIO, J. L Measles virus hemolysin. Virology 16: DENTON, J The pathology of fatal measles. Am. J. Med. Sci. 169: ENDERS, J. F., AND T. C. PEEBLES Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exptl. Biol. Med. 86: ENDERS, J. F Observations on certain viruses causing exanthematous diseases in man. Am. J. Med. Sci. 231: ENDERS, J. F., T. C. PEEBLES, K. MCCARTHY, M. MILOVANOVIC, A. Mrrus, AND A. HOLLO- WAY Measles virus: a summary of experiments concerned with isolation, properties, and behavior. Am. J. Public Health 47: ENDERS, J. F., K. MCCARTHY, A. MITUs, AND W. J. CHEATHAN Isolation of measles virus in cases of giant-cell pneumonia without rash. New Engl. J. Med. 261: ENDERS, J. F., S. L. KATZ, AND D. N. MEDEARIS, JR Recent advances in knowledge of the measles virus. Perspectives Virol. Symp., New York, p ENDERS, J. F., S. L. KATZ, M. V. MILOVANOVIC, AND A. HOLLOWAY Studies on an attenuated measles-virus vaccine. I. Development and preparation of the vaccine: technics for assay of effect of vaccination. New Engl. J. Med. 263: ENDERS, J. F Measles virus. Am. J. Diseases Children 103: FRANKL, J. W., T. BuRNSIEIN, J. T. DEVINEy, AND M. K. WEs.' Studies on isolates of measles virus. Bacteriol. Proc., p FRANKEL, J. W., T. BURNSTEIN, AND M. K. WEST Propagation of measles virus in

22 VOL. 30, 1966 MEASLES VIRUS IN CELL CULTURES 173 tissue cultures of dog kidney cells. Federation Proc. 17: FRANKEL, J. W., AND M. K. WEST Cultivation of measles virus in stable line of human amnion cells. Proc. Soc. Exptl. Biol. Med. 97: FRANKLIN, R. M., AND D. BALTIMORE Patterns of macromolecular synthesis in normal and virus-infected mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 27: FRIEDMAN, R. M Role of interferon in viral infection. Nature 201: FUNAHASHI, S., AND T. KATAWAKI Studies on measles virus hemagglutination. Biken's J. 6: GIFFORD, G. E., AND E. HELLER Effect of actinomycin D on interferon production by active and inactive Chikungunya virus in chick cells. Nature 200: GIRARDI, A. J., J. WARREN, C. GOLMAN, AND B. JEFFRIES Growth and CF antigenicity of measles virus in cells deriving from human heart. Proc. Soc. Exptl. Biol. Med. 98: GOLDBERG, I. H., AND M. RABINowrrz Actinomycin D inhibition of deoxyribonucleic acid-dependent synthesis of ribonucleic acid. Science 136: GOLDBERG, I. H., M. RABINowrrz, AND E. REICH Basis of actinomycin action. I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Natl. Acad. Sci. U.S. 48: GRESSER, I., AND S. L. KATZ Isolation of measles virus from urine. New Engl. J. Med. 263: HAMPARIAN, V. V., M. R. HILLEMAN, AND A. KELLER Contribution to characterization and classification of animal viruses. Proc. Soc. Exptl. Biol. Med. 112: HELLER, E Enhancement of Chikungunya virus replication and inhibition of interferon production by actinomycin D. Virology 21: Ho, M., AND J. F. ENDERS Further studies on inhibition of viral activity appearing in infected cell cultures and its role in chronic viral infections. Virology 9: Ho, M Interferons. New Engl. J. Med. 266: , , HOMMA, M., A. V. RAKE, W. PARANCHYCH, D. B. ELLIS, AND A. F. GRAHAM Biosynthess of viral ribonucleic acids, p In Viruses, nucleic acids, and cancer. The Williams & Wilkins Co., Baltimore. 53. HOSAKA, Y Morphology of the capsid of virus particles, p In Progress in Virology. Inst. Virus Res., Kyoto Univ. (in Japanese). 54. HOSAKA, Y Negative staining technique. J. Electron-microscopy (Tokyo) 13: HOWATSON, A. F Architecture and development of tumor viruses and their relation to viruses in general. Federation Proc. 21: HSIUNG, G. D., A. MANNINI, AND J. L. MELNICK Plaque assay of measles virus on Erythrocebus patas monkey kidney monolayers. Proc. Soc. Exptl. Biol. Med. 98: HUANG, C. H., P. Y. CHIA, F. T. CHU, K. C. Kuo, H. Y. WANG, T. L. Wu, AND H. H. Wu Studies on attenuated measles vaccine. I. Clinical and immunologic response to measles virus attenuated in human cells. Chin. Med. J. 81: HURWITZ, J., J. J. FURTH, M. MALAMY, AND M. ALEXANDER The role of deoxyribonucleic acid in ribonucleic acid synthesis. III. The inhibition of the enzymatic synthesis of ribonucleic acid and deoxyribonucleic acid by actinomycin D and proflavin. Proc. Natl. Acad. Sci. U.S. 48: IMAGAWA, D. T., AND J. M. ADAMS Propagation of measles virus in suckling mice. Proc. Soc. Exptl. Biol. Med. 98: INABA, Y., T. OMORI, M. KoNo, AND M. MATU- MOTO Parainfluenza 3 virus isolated from Japanese cattle. I. Isolation and identification. Japan. J. Exptl. Med. 33: JORDAN, W. S Human nasal cells in continuous culture. II. Virus susceptibilities. Proc. Soc. Exptl. Biol. Med. 92: KALLMAN, F., J. M. ADAMS, R. C. WILLIAMS, AND D. T. IMAGAWA Fine structure of cellular inclusions in measles virus infections. J. Biophys. Biochem. Cytol. 6: KAMAHORA, J Pathology of measles virus infection. Saishin-Igaku 19: KARAKI, T Studies on measles virus in tissue culture. I. Agar cell-suspension plaque assay of measles virus in stable cell lines. J. Japan. Assoc. Infect. Diseases 38: KARAKI, T Studies on measles virus in tissue culture. II. Cytochemical and fluorescent antibody studies on the growth of measles virus in FL cells. J. Japan. Assoc. Infect. Diseases 38: KARZON, D. T Measles virus. Ann. N.Y. Acad. Sci. 101: KATZ, S. L., M. V. MILOVANOVIC, AND J. F. ENDERS Propagation of measles virus in cultures of chick embryo cells. Proc. Soc. Exptl. Biol. Med. 97: KITAWAKI, T., S. FUNAHASHI, AND K. ToYo- SHIMA Purification and some antigenic properties of measles virus hemagglutinin. Biken's J. 6: KOHN, A Haemadsorption by measles syncytia. Nature 193: KOHN, A., AND D. YASSKY Growth of measles virus in KB cells. Virology 17: LAM, K. S. K., AND J. G. ATHERTON Measles virus. Nature 197: MASCOLI, C. C., L. V. STANFIELD, AND L. N. PHELPS Propagation of poliovirus, measles virus, and vaccinia in guinea pig spleen cel strains. Science 129: MATUMOTO, M., M. MUTAI, H. OGIWARA, I. NISHI, N. KUSANO, AND Y. AOYAMA

23 174 MATUMOTO BACTERIOL. REV. Isolement du virus de la rougeole en culture du tissue renal du singe. Compt. Rend. Soc. Biol. 153: MATUMOTO, M., T. KUMAGAI, T. SHIMIZU, AND S. IKEDA A new in vitro method (END) for detection and measurement of hog cholera virus and its antibody by means of effect of HC virus on Newcastle disease virus in swine tissue culture. II. Some characteristics of END method. J. Immunol. 87: MATUMOTO, M., M. MUTAI, AND H. OGIWARA Proliferation du virus rougeoleux en culture de cellules renales bovines. Compt. Rend. Soc. Biol. 155: MATUMOTO, M., M. MUTA:, Y. SABURI, R. FunII, M. MINAMITANI, AND K. NAKAMURA Live measles-virus vaccine: clinical trial of vaccine prepared from a variant of the Sugiyama strain adapted to bovine kidney cells. Japan. J. Exptl. Med. 32: MATUMOTO, M., Y. SABURI, Y. AOYAMA, AND M. MUTAL A neurotropic variant of measles virus in suckling mice. Arch. Ges. Virusforsch. 14: a.MATuMoTo, M., M. ARITA, AND M. ODA Enhancement of measles virus replication by actinomycin D. Japan. J. Exptl. Med. 35: MCCARTHY, K Measles. Brit. Med. Bull. 15: MCCARTHY, K Measles in laboratory hosts and tissue culture systems. Am. J. Diseases Children 103: MCCRUMB, F. R., JR., S. KRESS, E. SAUDERS, M. J. SNYDER, AND A. E. SCHLUEDERBERG Studies with live attenuated measlesvirus vaccine. I. Clinical and immunologic response in institutionalized children. Am. J. Diseases Children 101: MEYER, H. M., B. E. BROOKS, R. D. DOUGLAS, AND N. G. ROGERS Potency testing of live measles vaccine. Am. J. Diseases Children 103: MILOVANOVIC, M. V., J. F. ENDERS, AND A. Mrrus Cultivation of measles virus in human amnion cells and in developing chick embryo. Proc. Soc. Exptl. Biol. Med. 95: MUSSER, S. J., AND G. E. UNDERWOOD Studies on measles virus. II. Physical properties and inactivation studies of measles virus. J. Immunol. 85: MUSSER, S. J., AND E. A. SLATER Measles virus growth in canine renal cell cultures. Am. J. Diseases Children 103: MUTAI, M., Isolation and identification of measles virus. Japan. J. Exptl. Med. 29: NAGAHAMA, H., K. NAKAMURA, M. MINAMITANI, R. FuJiI, A. KAWAMURA, AND T. KAWASHIMA Application of fluorescent antibody technique to measles. Virus (Japan) 13: NISHI, Y., S. FUNAHASHI, T. KITAKAWA, AND K. FUKAI Micromorphological changes in measles infected KB cells. Biken's J. 5: NORRBY, E Hemagglutination by measles virus. III. Identification of two different hemagglutinins. Virology 19: NORRBY, E., B. FRIDING, G. ROCKBORN, AND S. GARD The ultrastructure of canine distemper virus. Arch. Ges. Virusforsch. 13: NORRBY, E Separation of measles virus components by equilibrium centrifugation in CsCl gradients. I. Crude and Tween and ether treated concentrated tissue culture material. Arch. Ges. Virusforsch 14: NORRBY, E. C. J., P. MAGNUSSON, L. G. FALKS- VEDEN, AND M. GRONBERG Separation of measles virus components by equilibrium centrifugation in CsCl gradients. II. Studies on the large and the small hemagglutinin. Arch. Ges. Virusforsch. 14: NORRBY, E. C. J., AND L. G. FALKSVEDEN Some general properties of the measles virus hemolysin. Arch. Ges. Virusforsch. 14: ODDO, F. G., R. FLANCCOMIO, AND A. SINATRA "Giant-cell" and "strand-forming" cytopathic effect of measles virus lines conditioned by serial propagation with diluted or concentrated inoculum. Virology 13: OKUNO, Y., T. SUGAI, T. FUJITA, T. YAMAMURA, K. TOYOSHIMA, M. TAKAHASHI, K. NAKAMURA, AND N. KUNITA Studies on the prophylaxis of measles with attenuated living virus. II. Cultivation of measles virus isolated by tissue culture in developing chick egg. Biken's J. 3: PERIEs, J. R., AND C. CHANY Activite hemagglutinante et hemolytique du virus morbilleux. Compt. Rend. 251: PERIES, J. R., AND C. CHANY Studies on measles viral hemagglutination. Proc. Soc. Exptl. Biol. Med. 110: PLOWRIGHT, W., J. G. CRUICKSHANK, AND A. P. WATERSON The morphology of rinderpest virus. Virology 17: RAPP, F., AND I. GORDON Development and spread of measles virus infection in human cells. Bacteriol. Proc., p RAPP, F., S. J. SELIGMAN, L. B. JAROSS, AND I. GORDON Quantitative determination of infectious units of measles virus by counts of immunofluorescent foci. Proc. Soc. Exptl. Biol. Med. 101: RAPP, F Observations of measles virus infection of human cells. III. Correlation of properties of clones of Hep-2 cells with their susceptibility to infection. Virology 10: RAPP, F., I. GORDON, AND R. F. BAKER Observation of measles virus infection of cultured human cells. I. A study of development and spread of virus antigen by means of immunofluorescence. J. Biophys. Biochem. Cytol. 7:43-48.

24 VOL. 30, 1966 MEASLES VIRUS INI CELL CULTURES REICH, E., R. M. FRANKLIN, A. J. SHATKIN, AND E. L. TATruM Effect of actinomycin D on cellular nucleic acid synthesis and virus production. Science 134: REICH, E., R. M. FRANKLIN, A. J. SHATKIN, AND E. L. TATUM Action of actinomycin D on animal cells and viruses. Proc. Natl. Acad. Sci. U.S. 48: REISINGER, R. C., K. L. HEDDLESTON, AND C. A. MANTHEI A myxovirus (SF-4) associated with shipping fever of cattle. J. Am. Vet. Med. Assoc. 135: REISSIG, M., F. L. BLACK, AND J. L. MELNICK Formation of multinucleated giant cells in measles virus infected cultures deprived of glutamine. Virology 2: REISSIG, M Electron microscopic study of the cytopathic changes induced by measles virus. Federation Proc. 17: ROIZMAN, B., AND A. E. SCHLUEDERBERG Virus infection of cells in mitosis. II. Measles virus infection of mitotic Hep-2 cells. Proc. Soc. Exptl. Biol. Med. 106: ROIZMAN, B The programing of herpes virus multiplication in mammalian cells, p In Viruses, nucleic acids, and cancer. The Williams & Wilkins Co., Baltimore ROSANOFF, E. I Hemagglutination and hemadsorption of measles virus. Proc. Soc. Exptl. Biol. Med. 106: ROSEN, L Hemagglutination and hemagglutination-inhibition with measles virus. Virology 13: RUCKLE, G Studies with measles virus. I. Propagation in different tissue culture systems. J. Immunol. 78: RUCKLE, G., AND K. D. ROGERS Studies with measles virus. II. Isolation of virus and immunologic studies in persons who have had the natural disease. J. Immunol. 78: RUCKLE, G Studies with measles virus. III. Attempts at isolation from post-mortem human tissue. J. Immunol. 79: RUCKLE, G Studies with the monkeyintra-nuclear-inclusion-agent (MINIA) and foamy-agent derived from spontaneously degenerating monkey kidney cultures. I. Isolation and tissue culture behavior of the agents and identification of MINIA as closely related to measles virus. Arch. Ges. Virusforsch. 8: RUCKLE, G Studies with the monkeyintra-nuclear-inclusion-agent (MINIA) and foamy-agent. II. Immunologic and epidemiologic observations in monkeys in a laboratory colony. Arch. Ges. Virusforsch. 8: RUCKLE-ENDERS, G Comparative studies of monkey and human measles-virus strains. Am. J. Diseases Children 103: RUSTIGIAN, R., P. JOHNSTON, AND H. REIHAST Infection of monkey kidney tissue cultures with virus-like agents. Proc. Soc. Exptl. Biol. Med. 88: RUSTIGIAN, R A carrier state in HeLa cells with measles virus (Edmonston strain) apparently associated with noninfectious virus. Virology 16: SABINA, L. R., AND R. J. WILSON Identification of attenuated strains. Am. J. Diseases Children 103: SABURI, Y., AND M. MATUMOTO Assay of measles virus hemolysin and its antibody. Arch. Ges. Virusforsch. 17: SATO, A Studies on measles. I. Susceptibility of various cell cultures to measles virus (Edmonston strain). Acta Paediat. Japon. 68: SCHLUEDERBERG, A. E Separation of measles virus particles in density gradients. Am. J. Diseases Children 103: SCHLUEDERBERG, A. E., AND B. ROIZMAN Separation of multiple antigenic components of measles virus by equilibrium sedimentation in cesium chloride. Virology 16: SCHWARZ, A. J. F., AND L. W. ZIRBEL Propagation of measles virus in non-primate tissue culture. I. Propagation in bovine kidney tissue culture. Proc. Soc. Exptl. Biol. Med. 102: SELIGMAN, S. J., AND F. RAPP A variant of measles virus in which giant cell formation appears to be genetically determined. Virology 9: SHINGU, M., AND Y. NAKAGAWA Studies on the measles virus. The isolation of measles virus on HeLa cells and immunological and morphological properties of the isolated agents. Kurume Med. J. 7: SMORODINTSEV, A. A., L. M. BOICHUK, AND E. S. SHIKINA Isolation attempts and investigations on measles virus strain. Works of the Pasteur Institute, Leningrad 17: TAWARA, J. T., J. R. GOODMAN, D. T. IMAGAWA, AND J. M. ADAMS Fine structure of cellular inclusions in experimental measles. Virology 14: TAWARA, J., M. YAMAMOTO, AND Y. MIZUHARA Sensitivity to measles virus infection of several cultured cells. Med. Biol. (Tokyo) 67: TAWARA, J Micromorphological changes in dog kidney cells infected with measles virus. Virus (Japan) 14: TAWARA, J Fine structure of filaments in dog kidney cell cultures infected with measles virus. Virology 25: TOYOSHIMA, K., M. TAKAHASHI, S. HATA, N. KUNITA, AND Y. OKUNO Virological studies on measles virus. I. Isolation of measles virus using FL cells and immunological properties of the isolated agents. Biken's J. 2: TOYOSHIMA, K., S. HATA, M. TAKAHASHI, N. KUNITA, AND Y. OKUNO Virological studies on measles virus. II. Growth of Toyoshima strain in four established cell lines. Biken's J. 2: