Download Persistent Infection of Vero Cells with Tacaribe Virus

J. gen. Virol. (1981), 56, 41-48. Printedin GreatBritain
41
Key words: Tacaribe virus/Vero cells~persistentinfection
Persistent Infection of Vero Cells with Tacaribe Virus
By E L S A B. D A M O N T E , * A N A C R I S T I N A D ' A I U T O L A AND
C E L I A E. C O T O
Cdtedra de Virologia, Departamento de Qu(mica Bioldgica, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabell6n 2, Piso 4 °, 1428,
Buenos Aires, Argentina
(Accepted 5 May 1981)
SUMMARY
Persistently infected cultures have been established from Vero cells surviving
primary infection with Tacaribe virus (Vero-T). The growth rate and morphological
characteristics of the persistently infected cells were indistinguishable from normal
Vero cells. Virus release declined during the first 6 passages, a cyclical pattern was
observed between passages 6 and 16, and subsequently no virus infectivity could be
detected. Co-cultivation with normal RK-13 or Vero cells enhanced virus yield from
virus-producing cultures of Vero-T cells (passage 15), but the addition of susceptible
cells had no effect on non-producer Vero-T cultures (passage 19). Only a small
proportion (< 1%) of the persistently infected cells tested during the first 16 passages
produced infectious virus. The virus released during the early stages of persistence
was temperature-sensitive if grown at 40 °C, more thermolabile at 50 °C than
parental virus, and unable to initiate a persistent infection in Vero cells. Vero-T cells
consistently showed refractoriness to homotypic Tacaribe virus superinfection and a
selective graded resistance to other arenavirus replication. The possible use of viral
susceptibility of persistently infected cultures as marker of antigenic relationship
among Tacaribe complex viruses is considered.
INTRODUCTION
The ability to persist in a rodent host or in cells Cultivated in vitro is a common property of
arenaviruses (Traub, 1939; Lehmann-Grube et al., 1969; Staneck et al., 1972; Dutko et al.,
1976; Sabattini et al., 1977; Coto et al., 1977). During the past few years studies in our
laboratory have been performed in order to chai'acterize persistent infection of Vero cells with
Junin virus (Coto et al., 1977). Not much is known about how the viral genome persists in
those cells, but different regulatory mechanisms such as generation of defective-interfering
(DI) particles (Help et al., 1976), host cell control through a DNA-dependent function (Coto
et al., 1979) or selection of thermosensitive (ts) mutants (Damonte & Coto, 1979a) have
been proposed to explain how carrier cultures are maintained.
The development of further studies with Junin virus was hampered by the lack of a good
plaquing system. Since we have found a highly reproducible plaque assay for Tacaribe virus
(TACV) in rabbit kidney cells (Damonte & Coto, 1979 b) we decided to continue our studies
in arenavirus persistence working with this virus. This paper describes the establishment and
characteristics of persistent infection of TACV in Vero cells. Several factors which may be
simultaneously involved in the regulation of virus persistence were analysed. The properties of
virus released during the early stage of persistence were studied in comparison with the
parental virus used to initiate the infection. Furthermore, we describe the properties of
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E.B.
DAMONTE~
A . C. D ' A I U T O L O
A N D C. E . C O T O
chronically infected cells, with particular emphasis on their responsiveness to superinfection
with Tacaribe complex viruses (Rowe et aL, 1970).
METHODS
Cell cultures. Monkey kidney Vero cells and rabbit kidney RK-13 cells were used. Both
cell lines were grown in Eagle's minimum essential medium (MEM) supplemented with 8 %
heat-inactivated calf serum and 50 #g/ml gentamicin, and maintained in Eagle's basal
medium (BME) containing 3 % calf serum and antibiotics.
Viruses. The TRLV 11573 strain of TACV used in this study was propagated in suckling
mouse brain and stocks were prepared as 10% brain homogenate. The arenaviruses Junin,
XJ.C13 strain, and Pichinde, Am 3739 strain, were propagated in suckling mouse brain. The
Indiana serotype of vesicular stomatitis virus (VSV) was subcultivated in BHK-21 cells.
Virus assays. Infectivity titrations of TACV, Pichinde and VSV were performed by plaque
assay in RK-13 cells at 37 °C as described elsewhere (Damonte & Coto, 1979b). Virus
plaques were counted 4, 6 and 8 days after infection by VSV, Pichinde and TACV
respectively. Junin virus was assayed in Vero cells (Coto et al., 1977).
Infectious centre assay. Monolayers of persistently infected cultures to be assayed were
detached from the bottles by trypsinization. The cell suspension was washed twice with BME,
resuspended in medium and counted in a haemocytometer. After adequate twofold step
dilutions, 0.2 ml amounts of the cell suspension were plated on to monolayers of RK- 13 cells
grown in 60 ml bottles. The cultures were incubated at 37 °C for 2 h and then carefully
overlaid with medium containing 1% agar. The number of plaque-forming cells was counted
8 days after staining the monolayers with 1% crystal violet.
Co-cultivation. Mixtures of approx. 106 persistently infected cells and 106 normal
uninfected RK-13 or Vero cells were seeded in 60 ml bottles, which were incubated at 37 or
40 °C. Supernatants from each mixed culture were harvested at 24, 48 and 72 h after seeding
and assayed for infectious TACV by plaque formation at 37 °C.
Heat inactivation. Four ml of the appropriate TACV sample diluted to contain approx. 104
p.f.u, was placed in a water bath at 50 °C. At 0, 10, 20, 30, 45 and 60 min, 0.5 ml samples
were removed. These samples were stored at 4 °C until all had been collected, at which time
they were assayed for remaining infectious TACV in RK-13 cells.
Viral interference assay. Vero cell cultures (106 cells/15 ml bottle) were inoculated either
with 0.2 ml of undiluted supernatants from Vero-T cells or with 0.2 ml BME. After a 1 h
adsorption period at 37 °C, monolayers were thoroughly washed with phosphate-buffered
saline (PBS) and re-infected with 106 p.f.u./culture of a standard stock of TACV. After a new
adsorption period and washing of the cells, cultures were fed with 3 ml BME. Twenty-four h
after infection, culture fluids were sampled and titrated for TACV on RK-13 monolayers.
RESULTS
Establishment of Vero cells persistently infected with TA C V
Chronically infected cultures of Vero cells (Vero-T) were established by infecting
monolayers with TACV at an input m.o.i, of 0-02. Virus was removed after a 1 h adsorption
period and cultures were fed with fresh medium.
At 4 days after infection, cultures exhibited marked cytopathic effects (c.p.e.) consisting of
cell rounding and detachment, and 90 % of the cells were destroyed 7 days later. By frequent
medium changes, regrowth of surviving cells was attained, and 19 days after the infection was
initiated cells were transferred by trypsinization. Vero-T cells were subcultured thereafter at
weekly intervals (split ratio 1:2) and monolayers were generally formed 2 to 3 days after
each passage. The Vero-T cell line was studied over a period of 200 days.
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Persistent infection with Tacaribe virus
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Fig. 1
Fig. 2
Fig. 1. Virus production in a culture of Veto cells persistently infected with TACV (Vero-T). Samples
of medium were taken at 3 to 4 days after each cell transfer and assayed for infectious virus on RK-13
cells.
Fig. 2. Inactivation kinetics of wild-type TACV and supernatants from Vero-T cell passages 2 and 3 at
50 °C. Samples containing approx. 104 p.f.u, were suspended in BME and heated in a 50 °C water
bath. At the indicated times, samples were withdrawn, rapidly chilled and assayed for surviving p.f.u.
The graphs were fitted by the least squares method. O, Wild-type TACV; D, supernatant from
Vero-T2; O, supernatant from Vero-T3.
The growth rate and morphological characteristics of the infected cell line were
indistinguishable from normal Vero cells. In addition, no spontaneous c.p.e, was observed in
the cultures. Similar observations have been recorded for other arenavirus persistently
infected lines previously studied (Damonte & Coto, 1979a; D a m o n t e et al., 1979; Dutko
et al., 1976).
Approx. 10% of Vero-T cells showed specific immunofluorescent staining.
Release of TA CV from persistently infected cultures
Three or 4 days after each cell transfer, supernatants from Vero-T cells were harvested and
titrated in order to measure spontaneous virus release (Fig. 1). During the first four passages,
virus levels in the medium ranged from 1.5 x 104 to 4.0 x 102 p.f.u./ml. Between passages 6
and 15, the production of infectious virus was greatly reduced and followed a cyclical pattern.
F r o m passage 16 onwards virus infectivity in Vero-T cell supernatants was not detected.
The low levels of virus released from Vero-T cells could be correlated with the proportion
o f virus producer cells. Analysis of the persistently infected cultures at different passage levels
showed that the percentage of Vero-T cells scoring as infectious centres varied from 1 to
0.0001%.
Properties of virus isolated from Vero-T cells; selection of ts mutants
The efficiency of replication of virus released from Vero-T cultures at different
temperatures was analysed in order to investigate the possible emergence of T A C V ts
mutants as described for the Vero cell-Junin virus system. RK-13 monolayers were infected
with supernatants from different passage levels of Vero-T cells. After virus adsorption for 1 h
at 37 °C, the cultures were washed to remove unadsorbed virus, fresh medium was added,
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E . B . D A M O N T E , A. C. D ' A I U T O L O A N D C. E. C O T O
Table 1. Temperature sensitivity of virus released from Vero-T cells at different passage
levels
Replication in RK- 13 cells
f
Virus yields (p.f.u./ml)*
)"
[
Cell passage
0 (5)~"
2 (37)
3 (44)
8 (65)
33 °C
4.9
1.4
5.2
2.3
×
×
×
×
104
104
102
103
•
40 °C/37 °C
37 °C
40 °C
ratio
1.1 × 105
6.5 × 104
1.7 x 104
4-9 × 103
1.7 × 10~
1.3 × 104
50
2.5
1.5
1 × 10-1
3 x 10-3
5 × 10-4
* Monolayers of RK-13 cells were infected with supernatants from different passages of Vero-T cells. After
virus adsorption for 1 h at 37 °C, the cultures were fed with fresh medium and incubated at 33 °C, 37 °C or 40 °C.
Four days after infection, culture fluids were sampled and titrated for TACV by plaque assay at 37 oC.
t Numbers in parentheses indicate days after initiation of Vero-T cell line.
and the cultures were incubated at 33, 37 or 40 °C. Four days after infection, culture fluids
were harvested and virus yields determined by plaque assay at 37 °C. Plaquing at 33 °C was
not attempted because T A C V produced very turbid (almost indistinguishable) plaques at this
temperature.
As shown in Table 1, fluids obtained from Vero-T cell cultures 5 to 65 days after initiation
of the persistent infection showed progressively reduced replicating efficiencies at the higher
temperature (40 °C). Thus, 5 days after infection, virus released from infected cells grew
equally well at all temperatures tested, but after 65 days a 2000-fold reduction in the yield at
40 °C over that found at 37 °C was registered. Since virus yields at 33 °C and 37 °C were
not significantly different the cut-off temperature for replication of the virus seems to be
above 37 °C. In all eases, 37 °C was the optimum growth temperature.
N o difference was observed in the characteristics of plaques produced by persistent ts
T A C V in comparison with parental T A C V nor was there an attenuation in the ability to kill
suckling mice as has been observed for other ts mutants isolated from viral carrier cultures
(Takemoto & Habel, 1959). In our case, the virulence for suckling mice of persistent and
parental T A C V measured as p.f.u./LDso ratio was similar: as few as 1 p.f.u, killed 5 0 % of the
inoculated mice, within an observation period of 20 days.
Further study of the virus isolated from Vero-T cell supernatants showed that persistent
virus was much more thermolabile than the parental one. As shown in Fig. 2, the infectivity of
virus isolated from Vero-T cell passages 2 and 3 was inactivated after heating at 50 °C more
rapidly than that o f parental TACV. The slope of the viral inactivation curve (Fig. 2) showed
a more rapid decline for virus released from Vero-T cells at passage 3 (Vero-T3) in
comparison with passage 2 (Vero-T2), indicating that the thermolability of the persistent virus
increased as the infection proceeded. Thus, values of half-life at 50 °C were 13, 7 and 3 min
respectively.
These results indicate that a population of thermolabile ts viruses was spontaneously
selected and it gradually replaced wild-type virus.
Susceptibility of Vero-T cells to superinfection with homologous or heterologous viruses
Despite the decline in virus production with time (Fig. 1), Vero-T cells consistently showed
refractoriness to homotypic virus superinfection. Cultures of Vero-T cells superinfected with
T A C V , at low or high multiplicity, did not show any cytopathic effect, while the yield of
superinfecting virus was always suppressed.
By passage 20, when Vero-T cell cultures were not releasing infectious virus, they were
assayed for susceptibility to different viruses in comparison with uninfected Vero cells. Both
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Persistent infection with Tacaribe virus
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Fig. 3. Growth curves of homotypic, heterotypic and heterologous viruses in Vero and Vero-T cells.
Monolayer cultures of Vero and Vero-T cells (passage 20) were infected with (a) TACV, (b) Junin, (c)
Pichinde or (d) VSV at an input m.o.i, of 10, 1, 0-5 and 0.005 respectively. After virus adsorption for 1
h at 37 °C, the monolayers were washed, fresh medium was added and the cultures were incubated at
37 °C. At various intervals after infection, culture fluids were harvested and assayed for infectious
virus. Cells were washed, frozen and thawed twice, and disrupted cells were assayed for cell-associated
virus. O , Released virus; O , cell-associated virus; - - ,
Vero cells; - - - , Vero-T cells.
types of cultures were challenged with the parental TACV, the related arenaviruses Junin and
Pichinde or the unrelated VSV. The growth curves of these challenger viruses in Vero and
Vero-T cells are shown in Fig. 3. As expected, replication of homotypic TACV in Vero-T
cells was completely suppressed (Fig. 3 a) whereas, the behaviour of the heterotypic Junin and
Pichinde viruses showed a significant difference. The growth of Junin virus, which shares
antigens with TACV in neutralization tests (Weissenbacher et al., 1975/76), was inhibited in
Vero-T cells; the latent period was delayed and virus titres were reduced approx. 3 loglo units
at days 3 to 4 post-infection (Fig. 3 b). By contrast, Vero-T cells were more permissive for
Pichinde virus replication since the virus titres were only 1 loglo unit lower than those
obtained in Vero cells (Fig. 3 c). The yields of the heterologous VSV in both cell systems were
similar (Fig. 3 d ) . Two points emerged from this experiment. First, the complete
permissiveness of Vero-T cells to the heterologous VSV replication, along with the known
refractoriness of Vero cells to produce interferon (Desmyter et al., 1968), allowed us to
exclude the possible involvement of interferon as a factor in the maintenance of the persistent
state. Second, the susceptibility of Vero-T cells to various member~ of the Tacaribe complex
is directly relevant to the antigenic relationship between TACV and the superinfecting virus.
Only a very close antigenic relatedness between persistent and challenge virus (as it is with
Junin and TACV) has led to a significant reduction of re-infecting virus expression.
The specific resistance to superinfection associated with persistently infected cells might be
explained in different possible ways. For instance, virus-induced modifications of the cell
surface receptor sites could block TACV re-infection by failure of cells to adsorb virus. To
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E . B . D A M O N T E , A. C. D ' A | U T O L O AND C. E. COTO
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48
72
Time after seeding (h)
Fig. 4. Influenceof co-cultivation of Vero-T cells with normal RK-13 cells. Mixtures of 106 Vero-Tcells
and 106 RK-13 cells were seeded in bottles and incubated at 37 °C or 40 °C. Supernatants from each
culture were harvested at 24, 48 and 72 h after seeding and titrated. Cultures incubated at 40 °C:
•
• , Vero-T cells passage 15 plus RK-13 cells; A
A. Vero-T 15 cells only. Cultures incubated
at 37 °C: 0-------0, Vero-T cells passage 15 plus RK-13 cells; O
O, Vero-T 15 cells only;
O - - - Q , Vero-T cells passage 19 plus RK-13 cells;O - - - - O , Vero-T 19 cells only.
elucidate this point, l0 s p.f.u, of TACV were used to infect Vero and Vero-T cultures (105
cells) and after adsorption for 1 h at 37 °C, the remaining infectivity in the inoculum was
determined. The results showed that TACV was adsorbed to Vero-T cultures with an
efficiency not significantly different from that of adsorption to normal uninfected cultures (78
and 86 % of virus adsorbed in 1 h respectively). From these results, we can conclude that the
blocking in the replication of superinfecting TACV occurs at some stage after adsorption of
virus to cells.
The resistance of Vero-T cells to homologous superinfection might alternatively be
attributed to interference by ts virions or D I particles. Experiments were therefore carried out
to test interfering activity in undiluted supernatants taken from persistently infected cultures
at early or late passage levels. No evidence was obtained of the presence of significant
numbers of DI particles in the fluids harvested from ts virus-producing or non-virusproducing Vero-T cell cultures. Supernatants from passages 2, 4, 7 or 22 of Vero-T cells
failed to produce any interference with the replication of a standard stock of T A C V in Veto
cells: the 24 h virus yields of doubly infected cultures were similar to those cultures infected
with TACV alone.
Influence of addition of susceptible cells to Vero- T cultures
Attempts were made to induce or enhance virus production from Vero-T cells through the
addition of susceptible cells to the persistently infected cultures by co-cultivation with RK-13
or Vero cells. Vero-T cells at passage levels 15 and 19 and RK-13 cells were seeded in equal
proportions and co-cultivated at various temperatures. At specified times after seeding, the
culture media were assayed for infectivity. The results are shown in Fig. 4. The addition of
normal cells altered the pattern of virus release from Vero-T cultures. If the persistently
infected cells were in a productive stage (passage 15), co-cultivation increased virus
production slightly, whereas in non-co-cultivated Vero-T cells the release of virus
progressively declined.
However, non-producer Vero-T cultures (passage 19) remained in that state although they
were mixed with normal RK-13 or Vero cells. Therefore, the loss of infectious virus during the
course of the persistent infection can be explained by the progressive lack of susceptible cells
in the culture, but, aside from that, there might be other requirements to rescue virus when
cells are in a non-productive state.
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Persistent infection with Tacaribe virus
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The results in Fig. 4 also show that shift of Vero-T cells to 40 °C resulted in diminished
virus production. As soon as 48 h after the shift to 40 °C there was a complete suppression of
virus release from Vero-T cultures. Co-cultivation allows virus multiplication at the restrictive
temperature. However, virus yields at 40 °C were reduced 90 to 99% in comparison with
virus yields at 37 °C indicating the presence of ts mutants in the virus population.
Inability of TA C V ts mutants to establish persistent infection
Attempts were made to establish a persistent infection in Vero cells using ts virus mutants
derived from Vero-T ceils. For that purpose, virus isolated from Vero-T cell passage 2, 8 and
15 supernatants were used. Vero cell monolayers grown in 15 ml bottles were infected and
after an adsorption period of 1 h at 37 °C, inoculum was withdrawn, fresh medium was
added and the cultures were incubated at 37 °C. Infected cells displayed marked c.p.e, at day
4 post-infection, cell destruction increased as the infection proceeded and, although many
frequent medium changes were done, the cultures could not be recovered.
This experience was repeated with ts mutants isolated from different passages of Vero-T
cells and, consistently, this virus developed a lytic infection characterized by complete
destruction of the cell monolayer without repopulation of the cultures.
DISCUSSION
As long as experimental data have been accumulated, persistence with RNA viruses
appears to be a rather complex biological phenomenon where different factors interplay and
none in particular is responsible for the establishment and maintenance of persistent infection.
Arenaviruses are specially prone to originate persistent infection whether or not they are
cytopathic for host cells. Summarizing the results obtained by others with LCM virus
persistent infections, DI particles or slow virus variants were always involved (Pfau, 1977;
Jacobson et al., 1979). We have also demonstrated the presence of DI particles in Vero cells
persistently infected with Junin virus (Vero-J cells) during a non-productive stage (Help et al.,
1976). Later work with another Vero-J cell line showed that infectious virus released, at an
early productive stage, was temperature-sensitive (Damonte & Coto, 1979 a) posing a role for
ts mutants in arenavirus persistence.
Data presented here demonstrate that mutants with a ts phenotype were also selected in
Vero-T cells. While this work was in progress, a rapid screening of the properties of virus
released by two additional lines of Vero-T cells confirmed the presence of ts mutants (Coto et
al., 1981), indicating that the generation of ts mutants in Veto cells persistently infected with
an arenavirus is a regular event. A peculiar feature is that all ts isolates were thermolabile at
50 °C, suggesting the possible importance of a structural protein alteration in regulatory
mechanisms of viral persistence.
Failure to detect DI particles in the supernatants of Vero-T cells cannot prove that they are
really absent since the parental virus used to establish the persistent infection contained DI
particles (Help & Coto, t980). Gimenez & Compans (1980) reported interfering activity due
to DI particles in supernatants of Vero cells persistently infected with TACV. However, virus
interference was demonstrated only after the supernatants had been concentrated 100-fold,
whereas we have used the supernatants as such.
Characteristically, production of virus progressively declined after several Vero-T cell
transfers and cultures displayed a total refractoriness to homotypic virus superinfection. A
progressive disappearance of permissive cells must occur since co-cultivation with normal
Vero cells slightly increases the level of virus synthesis. Alternatively, it could be assumed that
genetically resistant virus-free cells were selected. This seems to be unlikely because Vero-T
cells remained susceptible to the replication of Pichinde virus, suggesting that the cells
contained viral information essential for homotypic interference.
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E . B. D A M O N T E ,
A. C. D ' A I U T O L O
A N D C. E. C O T O
H o m o t y p i c e x c l u s i o n is a c o m m o n p r o p e r t y o f cells persistently infected with a r e n a v i r u s e s
( S t a n e c k et al., 1972; D a m o n t e & C o t o , 1979a). Besides, the results r e p o r t e d here s h o w that
the g r o w t h o f J u n i n virus was also restricted in V e r o - T cells, as if the susceptibility o f the
carrier culture to the superinfecting virus c o u l d be related to the degree o f antigenic
relatedness o f the virus carried by the cells.
This selective r e s p o n s e leads us to p r o p o s e the possible use o f the susceptibility o f
persistently infected cultures as a n o t h e r index o f antigenic relatedness o f m e m b e r s o f the
Tacaribe complex.
This work was supported by grants from the Secretaria de Ciencia y Tecnologia and Secretaria de
Salud Pfiblica, Argentina. E. B. D. and C. E. C. are Career Research members, Consejo Nacional de
Investigaciones Cienfificas y TOcnicas (Conicet), Argentina.
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(Received 15 January 1981)
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