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Interferon-mediated Persistent Infection of Saint Louis Encephalitis


									J. gen. Virol. (1982), 61, 177-186.   Printed in Great Britain                                 177

Key words: SLE/persistent infection~interferon~reptilian cell line

Interferon-mediated Persistent Infection of Saint Louis Encephalitis Virus
                        in a Reptilian Cell Line

                     By J. H. M A T H E W S *             AND A . V. V O R N D A M
  Vector-Borne Viral Diseases Division, Center for Infectious Diseases, Centers for Disease
  Control, Public Health Service, U.S. Department of Health and Human Services P.O. Box
                         2087, Fort Collins, Colorado 80522, U,S.A.

                                          (Accepted 31 March 1982)

        A persistent infection with Saint Louis encephalitis (SLE) virus in a poikilothermic
     cell line TH-1 (turtle heart cells) was studied. Infected TH-1 cells were subcultured
     weekly at 31 °C for 1 year and continued to produce low levels (102 to 10~ p.f.u./ml)
     of virus without obvious cytopathic effects or marked cyclic events. Indirect
     fluorescent antibody and infectious centre assays indicated that <1% of the cells
     were producing detectable virus proteins or infectious virus. Defective-interfering
     particles, temperature-sensitive mutants and DNA provirus were not detected.
     Interferon (IFN) mediation of the persistent infection was considered since the
     persistently infected cells (PIC) and normal TH- 1 cells were resistant to heterologous
     virus challenge after treatment with virus-free culture fluid from PIC. A direct
     relationship was found between the m,o.i, and the amount of IFN produced,
     plateauing at an m.o.i, of approx. 10. The reptilian IFN was physically and
     chemically similar to mammalian and avian IFN. Certain biological markers of the
     SLE virus changed during the persistent infection. It was less virulent for mice,
     showed, distinct differences in cell culture host range and had increased thermal

   Since chronic disease states and slow virus infections that occur in humans and animals are
not well understood, the study of the nature and mechanisms of persistent virus infections
with normally cytolytic viruses in cell culture has received considerable attention (Friedman
& Ramseur, 1979). Viruses from the Togaviridae and Rhabdoviridae families have been used
to study the persistent state in cell cultures of mammalian and arthropod origin. Examples of
such infections are vesicular stomatitis virus (VSV) in LY and BHK cells (Ramseur &
Friedman, 1977; Holland & Villarreal, 1974), rabies and Sindbis viruses in BHK and Aedes
albopictus cells (Kawai et al., 1975; Weiss et al., 1980; Riedel & Brown, 1977), Semliki
Forest virus (SFV) in mouse L929 cells and Japanese B virus in Vero and MA-111 cells
(Meinkoth & Kennedy, 1980; Schmaljohn & Blair, 1977).
   The establishment and maintenance of the persistent state may involve: (i) defective-
interfering (DI) particles (Huang & Baltimore, 1970; Grahm, 1977); (ii) temperature-
sensitive (ts) mutants (Igarashi et al., 1977; Preble & Youngner, 1975); (iii) interferon (IFN)
(Sekellick & Marcus, 1980; Friedman, 1977); (iv) integration as a provirus into the host
genome via activity of endogenous RNA-dependent DNA polymerase activity (Zhdanov &
Parfanovich, 1974; Haase et al., 1977). These factors may act in concert; DI particles and ts
mutants may be involved in establishing the persistent state, but IFN is responsible for the
long-term maintenance of the infection (Meinkoth & Kennedy, 1980).

178                     J. H. MATHEWS       AND    A . V. V O R N D A M

   Most persistent virus infections are studied in cells of homeothermic origin; poikilothermic
cells have not been well-utilized despite their ability to produce IFN and to support the
replication of a variety of viruses (Galabov et al., 1973; Falcoff & Fauconnier, 1965). A
chronic infection of rabies virus in viper cells has been described by Wiktor & Clark (1972),
but IFN was not detected. Likewise, a persistent fish rhabdovirus infection in Chinook
salmon embryo cells was not mediated by IFN (Engelking & Leong, 1981). Clark & Karzon
(1967a, b) developed a continuous cell line (TH-1) derived from the heart of a box turtle
(Terrapene carolina), which supports the replication of vaccinia virus, herpes simplex virus,
pseudorabies virus and VSV. In this report we describe a prolonged persistent infection of the
TH-1 cells with an arbovirus, Saint Louis encephalitis (SLE) virus. This persistent infection
was mediated by IFN, and biological changes in the virus were observed.

   Cells, viruses and immune sera. TH-1 cells were obtained from Dr H. F. Clark. They were
routinely grown at 31 °C in Eagle's minimum essential medium (MEM, Gibco) supplemented
with 10% (v/v) foetal calf serum (FCS), and subcultured weekly with a l :3 split ratio.
Primary duck embryo cell culture (DECC) and Vero cells were grown at 37 °C in the same
medium with 8% and 5% FCS respectively. Virus stock of a virulent strain of SLE virus
(MSI-7) (Monath et al., 1980) was prepared in DECC from the third suckling mouse brain
passage. VSV (Indiana) and Sindbis virus (Ar 339) were also grown in DECC. Hyperim-
mune ascitic fluid (HIAF) to SLE was obtained from the Arbovirus Reference Branch of this
   Plaque and infectious centre assays. All plaque assays were done in DECC. The agarose
overlay consisted of 0.5% agarose in MEM with 2% FCS, without phenol red dye. SLE
plaques were observed at 3 days without neutral red dye. Infectious centre assays with TH-I
cells were performed as described by Meinkoth & Kennedy (1980) but in DECC.
   Detection of virus proteins. An indirect fluorescent antibody (IFA) technique was used to
detect virus antigens in acetone-fixed TH-1 cells (Lyerla & Forrester, 1979). The fluorescein
conjugate (Miles-Yeda, Rehovot, Israel) was anti-mouse IgG prepared in rabbits.
   Interference. Interference of wild-type (wt) SLE virus replication by virus from
persistently infected cells (PIC) was determined in DECC and Vero cells. SLE virus from
PIC was concentrated with polyethylene glycol (PEG) 6000 (Union Carbide; 8 g/100 ml).
After 1 h at 4 °C, the precipitate was collected by centrifugation at 10000 g for 1 h and
resuspended in MEM. A standard concentration of wt SLE was mixed with varying amounts
of virus from PIC before inoculation. Culture fluids were sampled for virus at 72 h.
    Curing and cloning. The curing procedure used was similar to the one described by
Nishiyama (1977). PIC were passaged every 5 to 7 days into media with and without SLE
HIAF. The latter were checked for viruses after the cells had grown to confluency. TH-1 PIC
were cloned by terminal dilution in 96-well Linbro plates with 48 h conditioned MEM. After
the clones had grown to conftuency, the medium was checked for virus by plaque assay.
    Provirus induction. Inhibition of protein, RNA and DNA synthesis in cured and cloned
cells using 5-iodo-2'-deoxyuridine, cycloheximide, mitomycin C, and actinomycin D was
done according to the procedure of Schmaljohn & Blair (1979). After 48 h in the presence of
these drugs, the media and fluid from several rapid freeze-thaws of the cells were assayed for
virus in DECC. Normal DECC and Veto cells were co-cultivated with cured or cloned TH-1
 cells from PIC (1 : 1) at 31 °C and 39 °C. Culture fluids were assayed for virus at 6 days.
    Interferon assay. IFN activity was determined by inhibition of cytopathic effect (c.p.e.).
 Culture fluids were either acidified with 1 M-HCI to a pH of 2, stored overnight at 4 °C and
 then neutralized with 1 M-NaOH or were centrifuged at 40000 g for 6 h to pellet residual
 virus. Uninfected TH-1 cells, grown to confluency in 24-well Linbro culture plates, were
                                     Persistent infection o f Saint Louis virus                                               179
                                --   I    I     I   I   [ I\\1 I   I        I       I     I       I   I   I   I   I   I   I


                       ~ 4


                            l         1   I    I I I    I I,xl     I    I       II I I I I I
                                                                                      I       I
                                     1        3    5         7
                                                           "'2         6      14    18 22
                                                                                    10     26
                                              Days                           Weeks
                                                          Time post-infection
    Fig. 1. Replication of SLE (MSI-7) virus (m.o.i. of 1) in D E C C , Vero and TH- 1 cell cultures. The latter
    were then subcultured weekly and assayed for the presence of virus. O , D E C C ; A Vero; C], TH-1.

exposed overnight to 1 to 2 ml twofold dilutions of culture fluids. The media was removed;
cells were rinsed with phosphate-buffered saline (PBS) and were challenged with 100 TCIDs0
of VSV. The reciprocal of the last dilution not showing c.p.e, at 72 h was considered to be the
titre. IFN protection was also measured by reduction in virus yield after heterologous virus
challenge. VSV (105 TCID5o) was inoculated on to TH-1 PIC or normal cells that had been
treated with IFN culture fluids in 25 cm 2 tissue culture flats, adsorbed and washed thoroughly
with PBS; the media was sampled at 24 to 48 h and assayed for virus in DECC.
    Interferon characterization. IFN specimens without FCS were treated with 100/~g/ml
trypsin (Grand Island Biological, 1:300) at 37 °C for 1 h (Galabov et al., 1973). Trypsin
activity was stopped by adding FCS to a final concentration of 10%. IFN specimens were
also treated with RNase and DNase (Galabov et al., 1973b). As a control to ensure that
excess RNase had been used, RNase activity was detected by adding purified [3H]uridine-
labelled SLE RNA (Pedersen & Hazeltine, 1980) to an aliquot of supernatant fluid. After
incubation at 37 °C for 1 h, the RNA was centrifuged into a 15 to 30% sucrose gradient for
 18 h at 35 000 g. All of the virus RNA was degraded and remained at the top of the gradient.
IFN was treated with ether according to the procedure of Falcoff & Fauconnier (1965).
  Biological characterization of SLE virus from persistently infected TH-1 cells.
Three-week-old outbred Swiss mice were used for the SLE virus virulence studies. The mice
were inoculated intraperitoneally (i.p.) (10° to 105 p.f.u.) and observed for 2 weeks. Growth
curves of wt SLE and PIC viruses were done in DECC and Vero cells. Cells grown in 150
cm 2 flasks were inoculated with an m.o.i, of 0.001. Samples were taken over a 3 day period
for plaque assay. Thermal stability tests on viruses from PIC and wt ( 1 0 4 and 105 p.f.u./ml
respectively) were accomplished by diluting the virus suspensions into prewarmed MEM with
20% FCS and sampling periodically over 2 h. Controls at 4 °C were also included.


                                              Induction of the persistent state
   TH-1 cells are epithelial in nature but when adapted to grow at 31 °C the cells develop a
fusiform morphology and are not contact-inhibited (Clark & Karzon, 1967 b). SLE virus will
replicate in a variety of cell cultures. When growth curves with wt SLE were done in DECC,
180                     J. H. MATHEWS AND A. V. VORNDAM
Vero and T H - I cells, no c.p.e, and low virus titres were observed using the TH-1 cells;
however, both D E C C and Vero cells were lytically infected (Fig. 1). The replication pattern
of SLE virus in TH- 1 cells suggested that a persistent infection may have been established.
   Subsequent weekly subculturing of the TH-1 cells for an extended period of time showed
that infectious SLE virus levels in the culture supernatants remained between 102 and 10 4
p.f.u./ml. There were no marked virus cyclic events, and no c.p.e, has been observed.

               Evaluation of PIC for the production of virus or virus antigens
   Infectious centre assays (ICA) and IFA were used to determine the percentage of cells
expressing infectious virus or virus antigens. ICA showed that 25% of the cells contained
infectious virus 24 h after a primary infection, whereas only 0 . 2 % of the PIC at the 10th
passage level contained infectious SLE virus. It is known that cells can produce togavirus
antigens without producing infectious virus (Weiss et al., 1980; Schmaljohn & Blair, 1977),
but IFA showed that less than 1% of the PIC were expressing cytoplasmic virus antigens,
and only isolated positive cells were found. A solid-phase radioimmunoassay (Tew et al.,
 1977) confirmed that few PIC were expressing virus antigens (data not shown). Therefore, the
PIC did not harbour a detectable percentage of non-yielding cells.

                Influence of ts mutants and D l particles on the persistent state
   It is known that ts mutants can play a role in persistent infections; therefore, ICA and
routine plaquing techniques were performed with SLE virus from PIC and from culture fluids
at the commonly used permissive and restrictive temperatures of 31 °C and 39 °C. In all
cases the results from the plaque titrations of cells and fluids were the same at both
temperatures. These data indicate that ts viruses were not present in significant numbers.
   There was no evidence of a role for DI particles in this persistent infection, as is indicated
by the relatively low titres of virus produced without marked cyclic virus replication.
Nevertheless, we searched for DI particles by mixing in varying proportions of PEG-
concentrated SLE virus from PIC with the wt SLE. These were then inoculated on to DECC
and Vero cell cultures. In neither case did the presence of virus from PIC affect the final titre
of the wt SLE. The virus from the PIC did not replicate well in Vero cells.

                                   Cloning and curing of PIC
   Fifty cell clones from PIC were grown to confluency in 24 well Linbro cell culture plates.
In all cases the culture supernatants were negative for infectious SLE virus by plaque assay.
   It is characteristic of persistent infections that are maintained by horizontal transmission of
the virus that they can be 'cured' by ~passagin-g ~the,cells in the presence of antiviral sera
(Friedman & Ramseur, 1979). To determine if this was the case in TH-1 cells, PIC were
passaged in the presence of SLE HIAF. We found that by the sixth passage in the presence of
antibody the cultures no longer produced infectious virus as detected by plaque assay. When
the antibody was removed, the 'cured' cultures remained virus-free for six subsequent
passages. Parallel cultures, grown in the presence of normal HIAF, continued to replicate
SLE virus throughout the experiment.
   To determine if virus was still present in the cured or cloned cultures but not fully
expressed, the following tests were performed: (i) when the cells were tested by IFA for the
presence of virus antigens, the tests were uniformly negative; (ii) when the cells were tested for
heterologous interference by challenging them with VSV, the replication of the rhabdovirus
was not affected; (iii) when the cells were co-cultivated with Vero and DECC, no infectious
virus was produced; (iv) when the cells were treated with inhibitors of protein, RNA and
D N A synthesis to induce a possible provirus, no infectious virus was produced. These results
indicated that the cured or cloned PIC contained no inducible latent SLE virus.
                              Persistent infection o f Saint Louis virus                                      181
Table 1. Challenge of normal and S L E persistently infected TH-1 cells with homologous
                                           and heterologous virus

                                                  Virus titre at 48 h*
                  Cells             SLE          Sindbis         VSV         Control
             Persistent TH-lt       3-35           3-4           3-3          2-8
             TH-1                   4.6            6.3           5.9           -
      * Cells challenged at an m.o.i, of 1.
      "i"Culture fluids from PIC had an IFN titre of 1:4 by the c.p.e, method.
      $ Log~0p.f.u./ml. Control value represents the background SLE virus in persistent cell culture media.

                            Influence of interferon on the persistent state
    Normal TH-1 cells were treated for 24 h with culture fluids from various passages (10 to
30) of PIC and challenged with VSV. The yield of virus after 48 h was reduced by more than
90% in cells treated with culture media from PIC as compared to untreated cultures. This
indicated that a reptilian IFN-like substance was present in culture fluid from PIC.
    When I F N levels in culture fluids are low, Sekellick & Marcus (1980) suggest that
challenge of PIC may be more useful than assay of culture fluids as an indicator of I F N
activity. When our I F N titres were low, as measured by the c.p.e, method, the virus yield
from PIC after homologous wt or heterologous virus challenge was sharply reduced (Table
 1). These data indicated that an IFN-like substance was playing a role in the persistent
    Most studies on IFN induction show a typical dose-response curve until a plateau of 5 to
 10 m.o.i, is reached (Marcus et al., 1978; Fleischman & Simon, 1974). Other studies with
certain classes of DI particles from VSV and Sindbis show that maximum I F N production
occurs at lower m.o.i. (<1), higher m.o.i, drastically reducing IFN output (Sekellick &
Marcus, 1980). When culture fluids from wt SLE-challenged normal TH-1 cells were
evaluated over a 2-week period for I F N production by the c.p.e, method (Fig. 2 a, b), several
features could be observed. (i) The p.f.u./ml in the supernatant fluid on days 1 and 2 was
related to the initial m.o.i., i.e. higher m.o.i, produced higher virus titres (Fig. 2b). (ii) No
detectable virus was found in the higher m.o.i, sample by day 7 (Fig. 2 b). (iii) Higher m.o.i.
induced greater levels of I F N than the lower m.o.i. (Fig. 2a). (iv) The lower m.o.i, challenges
resulted in IFN production, but to lower titre; initial virus titres were reduced, but residual
virus was still present at day 12 (Fig. 2a, b). (v) Unchallenged PIC had maximum I F N
production on days 2 and 3 after subculturing. Virus titre was the highest on day 1, and then
decreased until day 8 when it remained constant (Fig. 2 a, b).
    Mammalian and avian I F N are characteristically resistant to or degraded by various
physical and chemical treatments (Bellanti, 1978). SLE-induced IFN from TH-1 cells
behaved similarly to other IFN (Table 2). TH-1 IFN was species-specific, since it would not
protect Vero or DECC from VSV challenge (data not shown).

                 Biological characterization of the SLE virus isolated from PIC
   The wt MSI-7 is a virulent strain of SLE virus (Monath et al., 1980). To determine if it had
undergone any changes in virulence during 35 passages, pooled culture fluids from various
passage levels were inoculated i.p. into 3-week-old mice (Fig. 3). After the third passage virus
from PIC was considerably less virulent than the wt. By the 35th passage, the LD~o had
increased approx. 10000-fold. Sera from animals inoculated with 100 p.f.u, of either the wt or
the 35th passage cultures were found to contain similar levels of neutralizing antibody (data
not shown).
182                               J, H. MATHEWS                        A N D A . V. V O R N D A M

                                          I            ~       I           I   I              I         I           I   1        l1

                    -~ 64

                     .~ 32

                          42      /                                                .... -" ---i-- ~~
                     <1:2'                                                                    i                 I            I
                          5       (b)

                     •        2
                                                                                          \                                       -


                         <1           I            I                   I         I                          I
                                      2       3            4       5      6      7                 8            9 10    11       12
                                                                       Time (days)
      Fig. 2. (a) Induction of IFN in TH 1 cells using various m.o.i.s of wt SLE virus. O, 100 : 1; ~, 10 : 1; [3,
       1: 1; O , 0-1 : 1; A, 1FN production in PIC after subculturing. (b) Levels of infectious SLE virus present
      in culture fluid of the challenged normal TH-I cells and the SLE PIC. For symbols, see (a).

         Table 2. Various physical and chemical treatments of TH-1 reptilian interferon

                                                       Treatment                     IFN titre*
                                              None                                                256
                                              56 ° C t                                             32
                                              Ether                                               128
                                              Trypsin$                                             16
                                              DNase and RNase§                                    256
                                              Dialysis                                            256

                                                  * As determined by the c.p.e, method.
                                                  $100/2g/ml, 1 h, 37°C.
                                                  § 100 #g/ml, 2 h, 37 °C, pH 7.
                                        Persistent infection of Saint Louis virus                                                      183
                                               I             I                       I           I       I        I           II
                                                                                                             IIIII 4b/l///

                      -~ 6 0 -

                      "~ 4 0 - _                                                  fl'-   ....
                                20 r                                  /

                                 0  5              4
                                                   .... I   A/

                                                                      3                      2       1

                                                       log~0 p.f.u, inoculated intraperitoneally
    Fig. 3. Virulence of SLE virus (wt) and virus taken post-infection and pooled at various passage (P)
    levels (i.e. 1 P = 7 days of the persistent infection) inoculated i.p. into 3-week-old Swiss mice. C), 3 P; G,
    8 to 10 P; v1, 15 to 16 P; O , 35 P; i , wt; n = 10 for wt and 35 P; n = 5 for the other samples.

                       100                 I            I                 I              I                                    I




                      <0. l
                                0         15           30             45                60                                   120
                                                                                     Time (rain)
    Fig. 4. Thermal stability at 48 °C for wt and SLE virus taken from PIC at 260 days post-infection. G,
    wt; O, SLE PIC.

   The decay curve for wt SLE is linear at a temperature of 48 °C. Thermal stability studies
at 48 °C showed that the SLE virus from PIC was more heat-labile than the wt SLE (Fig. 4).
   Growth curves of the two viruses were done in both DECC and Vero cell culture (Fig. 5).
We found that the virus from PIC did not replicate as well as the parent virus in DECC and
faiLd to replicate altogether in Vero cells.
   SLE wt virus in DECC routinely produces plaque sizes ranging from _<1 mm to 3 mm in
diam. The plaquing characteristics of the SLE virus from PIC and wt virus were similar.


   In the present study we have found that the persistent infection of SLE virus in a turtle
heart cell line was mediated by an interferon-like substance. Since the cells never went
through a virus-mediated crisis period, the TH-1 cells appeared to be persistently infected
from the time of infection. This infection met all the criteria set forth by Walker (1964) for a
virus-carrier culture mediated by IFN, including: (i) virus was cured from the culture with
184                                     J.   H.   MATHEWS                   AND           A.     V.       VORNDAM


                           4    5                 / //////                                                                            1


                                        ~ _ _ _F,.. _ _ _ _ . . 1 _ _ _ ~ _ _1._ _ _ _ ~           _ I. . . . .   ~ .....        f~
                                   10         20                  30         40                     50             60           70
                                                               T i m e p o s t - i n o c u l a t i o n (h)

      Fig. 5. G r o w t h c u r v e s o f w t (       ) a n d S L E v i r u s (m.o.i. o f 0 - 0 0 1 ) f r o m P I C ( - - - )   in D E C C   (O) and
      V e r o cells (A).

homologous virus antibody; (ii) clones from PIC or cured cells were not resistant to
heterologous challenge, whereas PIC were resistant to homologous and heterologous
challenge; (iii) only a small percentage of the cell population is infected or expressing virus
antigens when the carrier state is stable. Although factors other than IFN can make cells
refractory to heterologous challenge (Igarashi, 1979; Eaton, 1979), we found no ts mutants
or evidence for a putative interfering factor. Mediation of persistent virus infections with other
flaviviruses has involved DI particles with Japanese B encephalitis virus (Schmaljohn & Blaff,
1977) and IFN with tick-borne encephalitis virus (Stancek, 1965). Sekellick & Marcus (1980)
described the selection of certain classes of DI particles and small plaque ts mutants that
acted to maintain the persistent state. The role of DI particles and ts mutants in our study was
not apparent.
   Sekellick & Marcus (1980) have proposed a model for a regulatory role for IFN in
persistent virus infections through the phenomenon of cell sparing, where a delicate balance
arises between virus replication (cell killing) and virus inhibition (cell sparing). Our results
with SLE IFN induction in TH-1 cells support this model. The initial m.o.i, determined, in
general, the magnitude of the IFN response and whether or not the culture would be cured of
virus or develop a persistent-like state.
   SLE-induced IFN was species-specific and was physically and chemically similar to higher
phylum IFN. TH-1 IFN is heat-labile, but there is much diversity among IFN in susceptibility
to heat (Galabov et al., 1973).
   Biological variation in the SLE virus did occur. There were changes in virulence for mice,
thermal stability, and ability to replicate well in Vero cell culture. SLE viruses isolated from
nature are a biologically diverse group. Naturally occurring persistent infections such as we
have described may be a source for such variation.
   It is interesting to speculate whether such a persistent infection of reptiles might play some
role in the overwintering of arboviruses. Experimental inoculation of the alphaviruses
western equine encephalomyelitis virus and eastern equine encephalomyelitis virus into
snakes, with subsequent infection of mosquitoes after hibernation, has been reported by
Thomas & Ecklund (1960). However, Reeves (1974), in a review, concluded that the
evidence for the isolation of arboviruses in field circumstances, from poikilothermic
reservoirs, has been conflicting.
                                Persistent infection of Saint Louis virtts                                          185

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                                         (Received 3 December 1981)

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