Microbiol. Immunol. Vol. 21 (1), 23-31, 1977
Growth
of Measles Lymphoid Tomonori
Virus
in Various
Human
Cell Lines
MINAGAWA and
Takashi
SAKUMA
Departmentof Microbiology,Hokkaido UniversitySchoolof Medicine, Sapporo (Received for publication, July 28, 1976)
Abstract Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.
The replication of measles virus in human leukocytes in vitro was reported by Berg and Rosenthal (2). Recently, Sullivan et al (10) reported that measles virus could replicate in human lymphocytes of peripheral blood and furthermore Joseph et al (6) confirmed the replication of measles virus in B- and T-cells of human peripheral lymphocytes. Lymphoblastoid cell lines derived from B- and T-cells of human lymphocytes had been established and employed in studies of various fields. Chung and Murphy (3), and Gallagher and Flanagan (4) reported the replication of measles virus in human lymphoid cell lines of MOLT-4, Rajii and Jijoye. Joseph et al (6) and Barry et al (1) reported that measles virus of the wild type and attenuated Edmonstone strain could replicate and set up persistent infection in both B- and Tlymphoblastoid cells. Independently of their works, we also reported that the persistent infection of human lymphoid cell line of the B-cell type, NC-37, was established with an attenuated vaccine strain and a neurovirulent strain of measles virus and subacute sclerosing panencephalitis (SSPE) virus, but not with wild measles virus (8). The present report describes characteristics of the growth of attenuated measles virus, neurovirulent measles virus and SSPE virus in various human lymphoid cell lines of the T-cell type.
23
24
T.
MINAGAWA
AND T.
SAKUMA
MATERIALSAND METHODS Cell cultures. Human lymphoid cell lines of the T-cell type, MOLT-3, MOLT4 and CCRF-CEM cells, were used. These cell lines were kindly supplied by Dr. H. Katagiri, Department of Pathology, Asahikawa Medical College. These cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Vero cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 4% calf serum. Measlesviruses. Schwarz strain, vaccine lot no. H3-23 (1.4 x 104PFU/ml) which had been passaged serially in chick embryo cells was obtained commercially from Takeda Chem. Ind. Ltd. TYCSA strain which had been cloned from the wild Toyoshima strain (7) and shown to be highly neurovirulent in rodents (9) was supplied by Dr. K. Yamanouchi of National Institute of Health (1.2 x 106 PFU/ml). Halle strain of SSPE virus which had been isolated from lymph-node biopsy of a patient with SSPE by Horta-Barbosa et al (5) and undergone four passages in Vero cells was supplied by Dr. K. Yamanouchi (9.7 x 105 PFU/ml). Infection of cell cultures. One ml of human lymphoid cells (1 x 106 cells) was mixed with measles virus at an input multiplicity of 0.01. The mixtures were incubated at 37 C for 1 hr, centrifuged at 1,000 rmp for 5 min, and the cells were resuspended in 5 ml of fresh medium and cultivated in a plastic flask (Falcon 3013). Every day thereafter 1 ml was harvested from the culture and 1 ml of fresh medium was added during the initial 7 days, and subsequently harvesting 4 ml of culture fluid and replenishing with the same quantity of fresh medium was repeated with intervals of 5 to 7 days. The number of live cells and the percentage of hemadsorption-positive cells of each harvested sample was assayed using an aliquot while the residual samples were stored at -80 C for infectivity assay. Assay of infectionsvirus. Plaque assay for viral infectivity was performed in Vero cell monolayer cultures using 0.1 ml of virus inoculum per flask at an appropriate dilution. The procedures were described in a previous paper (8). Hamadsorption (Had) assay. Five-tenths ml of a cell suspension was mixed with 0.5 ml of 2% rhesus monkey erythrocytes, incubated at 37 C for 30 min, and the cells were washed 2 times with PBS to remove unadsorbed erythrocytes and then observed microscopically. Fluorescencemicroscopyof injected cells. Cells were smeared on a slide glass, airdried and fixed with cold acetone at 4 C for 30 min. They were exposed to fluorescein isothiocyanate (FITC)-labeled gamma-globulin of anti-measles rabbit serum (Toshiba Chem. Co.) at 37 C for 30 min, washed 3 times with PBS and observed microscoplically. Electron microscopy. Cells of carrier cultures were centrifuged at 1,000 rmp for 5 min. The cell pellet was fixed with 5% formaline for 16 hr and post-fixed with 2% osmium tetroxide for 2 hr, dehydrated with ethanol and acetone, and embedded in epoxyresin. After polymerization of the epoxy resin, sections were cut with an LKB ultramicrotome and stained with uranyl acetate followed by lead citrate. Electron microscopic observation was made in a Hitachi HS-9 electron microscope.
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
25
RESULTS
Growth Curves of Measles Virus in MOL T-3 Cells Schwarz, TYCSA and Halle strains were inoculated into MOLT-3 cells at an input multiplicity of 0.01. As displayed in Fig. 1, an infectious titer rise with Halle, TYCSA and Schwarz strains was detected on the 1st, 2nd and 7th day after inoculation, respectively. Almost all the cells in the cultures were shown to be infected, as demonstrated by hemadsorption and immunofluorescent staining, when the virus yield reached the maximum level. After that, the infected cells were degraded and the titers of infectious virus decreased. Cells infected with either Halle or TYCSA strain could never grow again but cells in the culture infected with Schwarz strain
Fig. 1. Growth curves of measles virus in MOLT-3 cells. MOLT-3 cells (1 x 106 cells) were inoculated with TYCSA (e●),Halle(○ (▲ 一 ▲)at
The
… ○)or
procedures
described
Schwarz
an input multiplicity
in the
of
infection
of
strain of O.Ol.
cells
arc
text.
began to grow again and the titer of infectious virus increased. Thus the replication of Schwarz strain repeated cyclically several times but finally all the cells were degraded on the 90th day after the first inoculation of the virus. GrowthCurvesof Measles Virus in MOL T-4 Cells As shown in Fig. 2, Halle, TYCSA and Schwarz strains could replicate in MOLT-4 cells and reached a maximum titer on the 4th, 5th and 9th day after inoculation, respectively, when almost all the cells were infected with these viruses. After that, all the cells were degraded.
26
T.
Fig.
MINAGAWA
2.
AND T.
Growth
MOLT-4
curves
cells)
cells. were
of
virus
cells
with
… ○)or
(▲--▲)at
measles
MOLT-4
inoculated
●),Halle(○
SAKUMA
in
(1 •~x 106
TYCSA
( •œ--
Schwarz
strain
an input multiplicity
of O.Ol.
Fig. 3. Growth curves of measles virus in CCRF-CEM cells. CCRF-CEM cells (1 x 106 cells) were inoculated with TYCSA (●--●), strain of
0.01.
Halle
( •£--•£
)
at
(○ … ○)or an
input
Schwarz multiplicity
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
27
Growth Curvesof Measles Virus in CCRF-CEM Cells With all the viral strains persistent infection was established in CCRF-CEM cells. As shown in Fig. 3, the virus yield of Halle, TYCSA and Schwarz strains reached a maximum titer on the 7th, 14th and 22nd day after inoculation, respectively. Thereafter, the infected cells were aggregated but not fused, grew at the same rate as unifected cells, and constantly produced infectious virus. Table
1.
Summary
of properties
of cells
persistently
infected
with
measles
virus
Propertiesof PersistentlyInfectedCCRF-CEM Cells Table 1 lists some properties of the cells persistently infected with TYCSA, Halle and Schwarz strains on the 100th day after virus inoculation. Almost all the cells were shown to be infected by hemadsorption and immuno-florescent staining. Capping phenomenon which had been observed in NC-37 cells persistently infected with TYCSA and Halle strains as reported previously (8), however, was not observed in CCRF-CEM cells persistently infected with those viruses. Cells infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titers of infectious virus, whereas cells infected with either TYCSA or Halle strain contained nucleocapsid structures only in the cytoplasm and produced high titers of infectious virus. Nucleocapsid structures with a diameter of 17 to 25 nm were seen only in the cytoplasm of cells persistently infected with these viruses and were not demarcated from the cytoplasmic matrix by a particular membrane (Figs. 4, 5, and 6). These structures were never seen in the nucleus. Nucleocapsid structures which were seen in the nucleus of cells persistently infected with Schwarz strain showed hollow structures with periodic striations on the outer surface and their width was uniform, measuring 14 to 16 nm (Fig. 4-B). DISCUSSION
The present study indicated that persistent infection of CCRF-CEM cells derived from T-cells of a human leukemia patient was established with attenuated measles virus of Schwarz strain, neurovirulent measles variant of TYCSA strain and Halle strain of SSPE virus. Previously we reported on the persistent infection of human lymphoid cells of the B-cell type (NC-37) with measles virus (8). Since the wild type Edmonston strain, the wild type Toyoshima strain and freshly isolated wild measles
28
T.
MINAGAWA
AND T.
SAKUMA
Fig. 4. Electron photomicrographs of a CCRF-CEM cell persistently infected with Schwarz strain. Tubular structures (arrows) in the nucleus and cytoplasm are seen (A). X9,000 High magnification ( x 72,000) of nucleocapsid structures in the nucleus (B).
GROWTH
OF
MEASLES
VIRUS
IN HUMAN
LYMPHOID
CELLS
Fig. 5. Electron photomicrograph of a CCRF-CEM cell persistently infected with Halle strain. Tubular structures (arrow) in the cytoplasm and particles budding on the cell surface are seen. x 9,000
Fig. 6. Electron photomicrograph of a CCRF-CEM cell persistently infected with TYCSA strain. Tubular structures (arrow) in the cytoplasm are seen. x 9,000
29
30
T.
MINAGAWA
AND T.
SAKUMA
virus could never establish persistent infection in NC-37 or CCRF-CEM cells (unpublished data), the attenuation and/or defectiveness of measles virus might be expressed by the establishment of persistent infection in either NC-37 or CCRFCEM cells. However, MOLT-3 and MOLT-4 cells were degraded by infection with these attenuated and/or defective viruses. Barry et al (1) also reported that the attenuated Edmonston strain could set up persistent infection in NC-37, Rajii and Foley cells but not in MOLT-4 and JiJoye cells. Thus it is conceivable that a cellular factor in addition to attenuation and/or defectiveness of virus strain might play a role in establishment of measles persistent infection. At present, the reason why these virus strains could not establish persistent infection in MOLT-3 and MOLT-4 cells is not clear. It might be due to the slow growth of cells or fragility of cells against infection with measles virus. Schwarz strain could be differentiated from either TYCSA strain or Halle strain by the analysis of growth curves of these viruses. The lag phase of Schwarz strain in various cell lines was usually longer than that of TYCSA or Halle strain. The growth pattern of TYCSA strain was similar to that of Halle strain. Such differentiation between virus strains was also demonstrated between the cells persistently infected with Schwarz strain and those infected with either TYCSA or Halle strain. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titers of infectious virus, whereas cells persistently infected with either TYCSA or Halle strain contained nucleocapsid structures only in the cytoplasm and produced high titers of infectious virus. These results agreed with the properties of measles persistent infection in NC-37 cells described in the previous paper (8). Capping phenomenon of measles antigens on the cell surface of NC-37 cells persistently infected with either TYCSA or Halle strain was demonstrated in the previous paper (8). However, in CCRF-CEM cells persistently infected with these viruses, capping of measles antigens could not be observed. This discrepancy might be due to the difference of source and nature of cells. Numerous microvilli were observed on the cell surface of NC-37 cells but not on CCRF-CEM cells. Capping of measles antigens in NC-37 cells followed cap-formation of microvilli. It was assumed that fluidity of the cytoplasmic membrane of NC-37 cells might be higher than that of CCRF-CEM cells. REFERENCES 1) Barry,D.W.,Sullivan,J.L., Lucas,S.J., Dunlap,R.C.,and Albrecht,P. 1976.Acuteand chronic infectionof human lymphoblastoid cell lineswithmeaslesvirus.J. Immunol.116:89-98. 2) Berg,R.B.,and Rosenthal,M.S. 1961.Propagationof measlesvirusin suspensions of humanand monkeyleukocytes.Proc. Soc.Exp. Biol.Med. 106:581-585. 3) Chung,M., and Murphy,W.H. 1970.Multiplicationof virusesin Burkittlymphomacells.J. Nat. CancerInst. 44: 1231-1239. 4) Gallagher,M.R., and Flanagan,T.D. 1976.Replicationofmeaslesvirusin continuouslymphoid cell lines.J. Immunol.116:1084-1088. 5) Horta-Barbosa,L., Hamilton,R., Wittig, B., Fuccillo,D.A., and Sever,J.L. 1971.Subacute sclerosing panencephalitis : Isolationof suppressedmeaslesvirusfromlymphnodebiopsies.Science 173:840-841.
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
31
6) Joseph, B.S., Lampert, P.W., and Oldstone, M.B.A. 1975. Replication and persistence of measles virus in defined subpopulation of human leukocytes. J. Virol. 16: 1638-1649. 7) Kohno, S., Kohase, M., and Suganuma, M. 1968. Growth of measles virus in a mouse derived established cell line L cell. Japan. J. Med. Sci. Biol. 21: 301-311. 8) Minagawa, T., Sakuma, T., Kuwajima, S., Yamamoto, T.K., and Iida, H. 1976. Characterization of measles viruses in establishment of persistent infections in human lymphoid cell line. J. Gen. Virol. 33: 361-379. 9) Shishido, A., Katow, S., Kobune, K., and Sato, T. 1973. Growth of measles virus in nervous tissues I. Neurotropic properties of measles virus in newborn hamsters. Japan. J. Med. Sci. Biol. 26: 103-118. 10) Sullivan, J.L., Barry, D.W., Lucas, S.J., and Albrecht, P. 1975. Measles infection of human mononuclear cells I. Acute infection of peripheral blood lymphocytes and monocytes. J. Exp. Med. 142: 773-784. Request for reprints should be addressed to Dr. T. Minagawa, Department Hokkaido University School of Medicine, Sapporo, Japan.
of Microbiology,
Growth
of Measles Lymphoid Tomonori
Virus
in Various
Human
Cell Lines
MINAGAWA and
Takashi
SAKUMA
Departmentof Microbiology,Hokkaido UniversitySchoolof Medicine, Sapporo (Received for publication, July 28, 1976)
Abstract Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.
The replication of measles virus in human leukocytes in vitro was reported by Berg and Rosenthal (2). Recently, Sullivan et al (10) reported that measles virus could replicate in human lymphocytes of peripheral blood and furthermore Joseph et al (6) confirmed the replication of measles virus in B- and T-cells of human peripheral lymphocytes. Lymphoblastoid cell lines derived from B- and T-cells of human lymphocytes had been established and employed in studies of various fields. Chung and Murphy (3), and Gallagher and Flanagan (4) reported the replication of measles virus in human lymphoid cell lines of MOLT-4, Rajii and Jijoye. Joseph et al (6) and Barry et al (1) reported that measles virus of the wild type and attenuated Edmonstone strain could replicate and set up persistent infection in both B- and Tlymphoblastoid cells. Independently of their works, we also reported that the persistent infection of human lymphoid cell line of the B-cell type, NC-37, was established with an attenuated vaccine strain and a neurovirulent strain of measles virus and subacute sclerosing panencephalitis (SSPE) virus, but not with wild measles virus (8). The present report describes characteristics of the growth of attenuated measles virus, neurovirulent measles virus and SSPE virus in various human lymphoid cell lines of the T-cell type.
23
24
T.
MINAGAWA
AND T.
SAKUMA
MATERIALSAND METHODS Cell cultures. Human lymphoid cell lines of the T-cell type, MOLT-3, MOLT4 and CCRF-CEM cells, were used. These cell lines were kindly supplied by Dr. H. Katagiri, Department of Pathology, Asahikawa Medical College. These cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Vero cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 4% calf serum. Measlesviruses. Schwarz strain, vaccine lot no. H3-23 (1.4 x 104PFU/ml) which had been passaged serially in chick embryo cells was obtained commercially from Takeda Chem. Ind. Ltd. TYCSA strain which had been cloned from the wild Toyoshima strain (7) and shown to be highly neurovirulent in rodents (9) was supplied by Dr. K. Yamanouchi of National Institute of Health (1.2 x 106 PFU/ml). Halle strain of SSPE virus which had been isolated from lymph-node biopsy of a patient with SSPE by Horta-Barbosa et al (5) and undergone four passages in Vero cells was supplied by Dr. K. Yamanouchi (9.7 x 105 PFU/ml). Infection of cell cultures. One ml of human lymphoid cells (1 x 106 cells) was mixed with measles virus at an input multiplicity of 0.01. The mixtures were incubated at 37 C for 1 hr, centrifuged at 1,000 rmp for 5 min, and the cells were resuspended in 5 ml of fresh medium and cultivated in a plastic flask (Falcon 3013). Every day thereafter 1 ml was harvested from the culture and 1 ml of fresh medium was added during the initial 7 days, and subsequently harvesting 4 ml of culture fluid and replenishing with the same quantity of fresh medium was repeated with intervals of 5 to 7 days. The number of live cells and the percentage of hemadsorption-positive cells of each harvested sample was assayed using an aliquot while the residual samples were stored at -80 C for infectivity assay. Assay of infectionsvirus. Plaque assay for viral infectivity was performed in Vero cell monolayer cultures using 0.1 ml of virus inoculum per flask at an appropriate dilution. The procedures were described in a previous paper (8). Hamadsorption (Had) assay. Five-tenths ml of a cell suspension was mixed with 0.5 ml of 2% rhesus monkey erythrocytes, incubated at 37 C for 30 min, and the cells were washed 2 times with PBS to remove unadsorbed erythrocytes and then observed microscopically. Fluorescencemicroscopyof injected cells. Cells were smeared on a slide glass, airdried and fixed with cold acetone at 4 C for 30 min. They were exposed to fluorescein isothiocyanate (FITC)-labeled gamma-globulin of anti-measles rabbit serum (Toshiba Chem. Co.) at 37 C for 30 min, washed 3 times with PBS and observed microscoplically. Electron microscopy. Cells of carrier cultures were centrifuged at 1,000 rmp for 5 min. The cell pellet was fixed with 5% formaline for 16 hr and post-fixed with 2% osmium tetroxide for 2 hr, dehydrated with ethanol and acetone, and embedded in epoxyresin. After polymerization of the epoxy resin, sections were cut with an LKB ultramicrotome and stained with uranyl acetate followed by lead citrate. Electron microscopic observation was made in a Hitachi HS-9 electron microscope.
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
25
RESULTS
Growth Curves of Measles Virus in MOL T-3 Cells Schwarz, TYCSA and Halle strains were inoculated into MOLT-3 cells at an input multiplicity of 0.01. As displayed in Fig. 1, an infectious titer rise with Halle, TYCSA and Schwarz strains was detected on the 1st, 2nd and 7th day after inoculation, respectively. Almost all the cells in the cultures were shown to be infected, as demonstrated by hemadsorption and immunofluorescent staining, when the virus yield reached the maximum level. After that, the infected cells were degraded and the titers of infectious virus decreased. Cells infected with either Halle or TYCSA strain could never grow again but cells in the culture infected with Schwarz strain
Fig. 1. Growth curves of measles virus in MOLT-3 cells. MOLT-3 cells (1 x 106 cells) were inoculated with TYCSA (e●),Halle(○ (▲ 一 ▲)at
The
… ○)or
procedures
described
Schwarz
an input multiplicity
in the
of
infection
of
strain of O.Ol.
cells
arc
text.
began to grow again and the titer of infectious virus increased. Thus the replication of Schwarz strain repeated cyclically several times but finally all the cells were degraded on the 90th day after the first inoculation of the virus. GrowthCurvesof Measles Virus in MOL T-4 Cells As shown in Fig. 2, Halle, TYCSA and Schwarz strains could replicate in MOLT-4 cells and reached a maximum titer on the 4th, 5th and 9th day after inoculation, respectively, when almost all the cells were infected with these viruses. After that, all the cells were degraded.
26
T.
Fig.
MINAGAWA
2.
AND T.
Growth
MOLT-4
curves
cells)
cells. were
of
virus
cells
with
… ○)or
(▲--▲)at
measles
MOLT-4
inoculated
●),Halle(○
SAKUMA
in
(1 •~x 106
TYCSA
( •œ--
Schwarz
strain
an input multiplicity
of O.Ol.
Fig. 3. Growth curves of measles virus in CCRF-CEM cells. CCRF-CEM cells (1 x 106 cells) were inoculated with TYCSA (●--●), strain of
0.01.
Halle
( •£--•£
)
at
(○ … ○)or an
input
Schwarz multiplicity
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
27
Growth Curvesof Measles Virus in CCRF-CEM Cells With all the viral strains persistent infection was established in CCRF-CEM cells. As shown in Fig. 3, the virus yield of Halle, TYCSA and Schwarz strains reached a maximum titer on the 7th, 14th and 22nd day after inoculation, respectively. Thereafter, the infected cells were aggregated but not fused, grew at the same rate as unifected cells, and constantly produced infectious virus. Table
1.
Summary
of properties
of cells
persistently
infected
with
measles
virus
Propertiesof PersistentlyInfectedCCRF-CEM Cells Table 1 lists some properties of the cells persistently infected with TYCSA, Halle and Schwarz strains on the 100th day after virus inoculation. Almost all the cells were shown to be infected by hemadsorption and immuno-florescent staining. Capping phenomenon which had been observed in NC-37 cells persistently infected with TYCSA and Halle strains as reported previously (8), however, was not observed in CCRF-CEM cells persistently infected with those viruses. Cells infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titers of infectious virus, whereas cells infected with either TYCSA or Halle strain contained nucleocapsid structures only in the cytoplasm and produced high titers of infectious virus. Nucleocapsid structures with a diameter of 17 to 25 nm were seen only in the cytoplasm of cells persistently infected with these viruses and were not demarcated from the cytoplasmic matrix by a particular membrane (Figs. 4, 5, and 6). These structures were never seen in the nucleus. Nucleocapsid structures which were seen in the nucleus of cells persistently infected with Schwarz strain showed hollow structures with periodic striations on the outer surface and their width was uniform, measuring 14 to 16 nm (Fig. 4-B). DISCUSSION
The present study indicated that persistent infection of CCRF-CEM cells derived from T-cells of a human leukemia patient was established with attenuated measles virus of Schwarz strain, neurovirulent measles variant of TYCSA strain and Halle strain of SSPE virus. Previously we reported on the persistent infection of human lymphoid cells of the B-cell type (NC-37) with measles virus (8). Since the wild type Edmonston strain, the wild type Toyoshima strain and freshly isolated wild measles
28
T.
MINAGAWA
AND T.
SAKUMA
Fig. 4. Electron photomicrographs of a CCRF-CEM cell persistently infected with Schwarz strain. Tubular structures (arrows) in the nucleus and cytoplasm are seen (A). X9,000 High magnification ( x 72,000) of nucleocapsid structures in the nucleus (B).
GROWTH
OF
MEASLES
VIRUS
IN HUMAN
LYMPHOID
CELLS
Fig. 5. Electron photomicrograph of a CCRF-CEM cell persistently infected with Halle strain. Tubular structures (arrow) in the cytoplasm and particles budding on the cell surface are seen. x 9,000
Fig. 6. Electron photomicrograph of a CCRF-CEM cell persistently infected with TYCSA strain. Tubular structures (arrow) in the cytoplasm are seen. x 9,000
29
30
T.
MINAGAWA
AND T.
SAKUMA
virus could never establish persistent infection in NC-37 or CCRF-CEM cells (unpublished data), the attenuation and/or defectiveness of measles virus might be expressed by the establishment of persistent infection in either NC-37 or CCRFCEM cells. However, MOLT-3 and MOLT-4 cells were degraded by infection with these attenuated and/or defective viruses. Barry et al (1) also reported that the attenuated Edmonston strain could set up persistent infection in NC-37, Rajii and Foley cells but not in MOLT-4 and JiJoye cells. Thus it is conceivable that a cellular factor in addition to attenuation and/or defectiveness of virus strain might play a role in establishment of measles persistent infection. At present, the reason why these virus strains could not establish persistent infection in MOLT-3 and MOLT-4 cells is not clear. It might be due to the slow growth of cells or fragility of cells against infection with measles virus. Schwarz strain could be differentiated from either TYCSA strain or Halle strain by the analysis of growth curves of these viruses. The lag phase of Schwarz strain in various cell lines was usually longer than that of TYCSA or Halle strain. The growth pattern of TYCSA strain was similar to that of Halle strain. Such differentiation between virus strains was also demonstrated between the cells persistently infected with Schwarz strain and those infected with either TYCSA or Halle strain. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titers of infectious virus, whereas cells persistently infected with either TYCSA or Halle strain contained nucleocapsid structures only in the cytoplasm and produced high titers of infectious virus. These results agreed with the properties of measles persistent infection in NC-37 cells described in the previous paper (8). Capping phenomenon of measles antigens on the cell surface of NC-37 cells persistently infected with either TYCSA or Halle strain was demonstrated in the previous paper (8). However, in CCRF-CEM cells persistently infected with these viruses, capping of measles antigens could not be observed. This discrepancy might be due to the difference of source and nature of cells. Numerous microvilli were observed on the cell surface of NC-37 cells but not on CCRF-CEM cells. Capping of measles antigens in NC-37 cells followed cap-formation of microvilli. It was assumed that fluidity of the cytoplasmic membrane of NC-37 cells might be higher than that of CCRF-CEM cells. REFERENCES 1) Barry,D.W.,Sullivan,J.L., Lucas,S.J., Dunlap,R.C.,and Albrecht,P. 1976.Acuteand chronic infectionof human lymphoblastoid cell lineswithmeaslesvirus.J. Immunol.116:89-98. 2) Berg,R.B.,and Rosenthal,M.S. 1961.Propagationof measlesvirusin suspensions of humanand monkeyleukocytes.Proc. Soc.Exp. Biol.Med. 106:581-585. 3) Chung,M., and Murphy,W.H. 1970.Multiplicationof virusesin Burkittlymphomacells.J. Nat. CancerInst. 44: 1231-1239. 4) Gallagher,M.R., and Flanagan,T.D. 1976.Replicationofmeaslesvirusin continuouslymphoid cell lines.J. Immunol.116:1084-1088. 5) Horta-Barbosa,L., Hamilton,R., Wittig, B., Fuccillo,D.A., and Sever,J.L. 1971.Subacute sclerosing panencephalitis : Isolationof suppressedmeaslesvirusfromlymphnodebiopsies.Science 173:840-841.
GROWTH
OF
MEASLES
VIRUS
IN
HUMAN
LYMPHOID
CELLS
31
6) Joseph, B.S., Lampert, P.W., and Oldstone, M.B.A. 1975. Replication and persistence of measles virus in defined subpopulation of human leukocytes. J. Virol. 16: 1638-1649. 7) Kohno, S., Kohase, M., and Suganuma, M. 1968. Growth of measles virus in a mouse derived established cell line L cell. Japan. J. Med. Sci. Biol. 21: 301-311. 8) Minagawa, T., Sakuma, T., Kuwajima, S., Yamamoto, T.K., and Iida, H. 1976. Characterization of measles viruses in establishment of persistent infections in human lymphoid cell line. J. Gen. Virol. 33: 361-379. 9) Shishido, A., Katow, S., Kobune, K., and Sato, T. 1973. Growth of measles virus in nervous tissues I. Neurotropic properties of measles virus in newborn hamsters. Japan. J. Med. Sci. Biol. 26: 103-118. 10) Sullivan, J.L., Barry, D.W., Lucas, S.J., and Albrecht, P. 1975. Measles infection of human mononuclear cells I. Acute infection of peripheral blood lymphocytes and monocytes. J. Exp. Med. 142: 773-784. Request for reprints should be addressed to Dr. T. Minagawa, Department Hokkaido University School of Medicine, Sapporo, Japan.
of Microbiology,
