J. gen. Virol. (1984), 65, 1909-1920.
1909
Printed in Great Britain
Key words: DI virus/SFV/inhibition o f multiplication
Protection of Mice Infected with a Lethal Dose of Semliki Forest Virus by
Defective Interfering Virus: Modulation of Virus Multiplication
By A. D. T. B A R R E T T , A. R. G U E S T , A. M A C K E N Z I E
N. J. D I M M O C K *
1 AND
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. and
1Institute for Research on Animal Diseases, Compton, Berkshire, RG16 ONN, U.K.
(Accepted 1 August 1984)
SUMMARY
Certain defective interfering (DI) Semliki Forest virus (SFV) preparations
completely protected the majority of mice inoculated with a normally lethal dose of
SFV, and the surviving mice showed no signs of disease during the period of
observation. Depending upon which DI SFV preparation was used, the survivors were
resistant to challenge with 100 LDs0 SFV (DI SFV pl3a), or were completely sensitive
(DI SFV p4), the latter having evidently failed to establish a protective immunity. In
this report we compared the ability of these two DI SFV preparations to inhibit multiplication of infectious virus in mice inoculated with 10 LDs0 SFV. The following conclusions emerged: (i) virus multiplication was profoundly inhibited in the majority of
mice treated with either of the DI virus preparations although there was significant
multiplication in most tissues, including brain. The number of mice showing evidence
of reduced infectivity titres (58 ~ ) correlated well with the 60 ~ which survived without
disease in lethality experiments. (ii) Despite the presence of infectivity, no SFV antigen
or histopathological lesions were detected in brain or spinal cord. (iii) The DI virus
preparations p4 and pl 3a altered the distribution of infectivity in the mouse in different ways: during the first 2 days of-the infection modulated by DI virus p4, the infectivity titres (in brain, olfactory lobes and spleen) were comparatively high, being > 1 ~ of
those in mice inoculated with standard virus alone. However, from day 3, titres declined precipitously and there was little infectivity in any of the tissues investigated. On
the other hand, mice treated with DI SFV pl 3a had, over the entire duration of infection, greatly reduced though significant infectivity in brain, olfactory lobes and spleen
and very little infectivity in serum. (iv) In a minority of mice (14.5~o), DI virus pl3a
altered the distribution of infectivity between different tissues so that there was
significantly decreased virus in just one or two of the four tissues investigated,
suggesting that the infection was being subtly modulated by the DI virus. (v)
Interference assays failed to detect DI SFV in any tissue samples although the effects of
DI virus on infection in the mouse were obvious.
INTRODUCTION
Defective interfering (DI) virus particles arise by the deletion of a major part of the genome of
standard (infectious) virus and as a result can only multiply in cells co-infected with standard
virus. Such co-infection results in a reduced yield of standard virus progeny, a phenomenon
know as homotypic interference. DI particles are spontaneously produced during serial passage
at both high (Perrault, 1981) and low (Barrett et al., 1981) multiplicity of infection of many, if not
all, animal viruses (Huang & Baltimore, 1977; Holland et al., 1980; Perrault, 1981) but because
of their dependence on standard virus, multiplication of DI virus is optimal at high m.o.i. Under
these conditions DI virus particles become enriched and can outnumber those of the standard
virus. Interference occurs intracellularly and is specific for the virus from which the DI virus was
derived (Perrault, 1981), although interference has been reported between standard virus and DI
virus belonging to different serotypes of vesicular stomatitis virus (VSV) (Prevec & Kang, 1970;
0022-I317/84/0000-6200 $02.00© 1984 SGM
1910
A.D.T.
BARRETT AND OTHERS
Schnitzlein & Reichmann, 1976) and between DI Semliki Forest virus (SFV) and Sindbis virus
(Barrett & Dimmock, 1984a).
To date, most of the work on DI particles has been concerned with the molecular biology (Perrault, 1981 ; Lazzarini et al., 198 l) rather than the modulation of infection by D I particles in vivo,
although this possibility was first suggested by Huang & Baltimore (1970). Several workers have
shown that animals can be protected from acute virus infections by exogenous application of D I
virus, for example with VSV inoculated intracerebrally (Doyle & Holland, 1973 ; Holland & Villareal, 1975; Rabinowitz et al., 1977; Jones & Holland, 1980) or intraperitoneally (Fultz et al.,
1982), with reovirus (Spandidos & Graham, 1976), with influenza virus (von Magnus, 1951 ; Rabinowitz & Huprikar, 1979), with lymphocytic choriomeningitis virus (Welsh et al., 1977) and
with SFV (Dimmock & Kennedy, 1978; Crouch et al., 1982; Barrett & Dimmock, 1984b, c).
However, only a few studies have used appropriate controls to demonstrate that these effects are
due to interference rather than to the immunogenic effects of D I virus particle antigens (Jones &
Holland, 1980; D i m m o c k & Kennedy, 1978 ; Crouch et al., 1982; Barrett & Dimmock, 1984b, c).
As a result of administering DI virus, the acute infection may become entirely subclinical (SFV:
D i m m o c k & Kennedy, 1978; Barrett & Dimmock, 1984 c) or converted to a chronic wasting condition (VSV inoculated intracerebrally: Doyle & Holland, 1973 ; reovirus: Spandidos & G r a h a m ,
1976), or a long-term persistent infection (VSV inoculated intraperitoneally: Fultz et al., 1982).
However, almost nothing is known of how virus and host interact to bring about profound
changes in the expression of disease.
In earlier reports we demonstrated that SFV DI virus can protect mice from a lethal SFV
encephalitis (Dimmock & Kennedy, 1978; Crouch et al., 1982). However, we subsequently
found that DI viruses are heterogeneous in their biological properties and we defined three categories: (1) DI virus which protected mice and left them i m m u n e to challenge at 3 weeks after
infection with 100 LD5o SFV; (2) DI virus which protected mice but failed to establish i m m u n ity to challenge; (3) DI virus which did not protect mice even though it had the same interference
titre in vitro as the other preparations (Barrett & Dimmock, 1984c). In this report we have examined the extent to which the first two categories of DI SFV modulate the multiplication of SFV
in various tissues of the mouse.
METHODS
Viruses. Virulent SFV (ts+; standard virus) was grown in BHK-21 cells (m.o.i. = 0.1) as described by Dimmock
& Kennedy (1978). Avirulent SFV was obtained by passage in BHK-21 cells (m.o.i. = 0.1) for 18 h at 37 °C from
the avirulent strain A774 (Bradish et al., 1971). DI SFV preparations were derived by serial undiluted passage of
SFV in BHK cells which in later passages were maintained at a constant m.o.i, of 50 p.f.u./cell by the addition of
infectious virus. Incubation was for 24 h at 37 °C. DI SFV p4 (i.e. four undiluted passages) was generated from
standard virus as described above, while DI SFV pl3a was derived from the DI SFV p8 used by Dimmock &
Kennedy (1978).
Cells. Chick embryo fibroblast (CEF) monolayes were prepared as described by Morser et al. (1973) and BHK21 and L929 cells were grown as monolayers by standard techniques.
Infectivity titrations. Virus was plaque assayed in CEF (Kennedy & Burke, 1972) at 33 °C, with an agar overlay
containing 0-02~0 DEAE-dextran.
Mice. Male CFLP random bred mice 4 to 5 weeks old weighing about 25 g were obtained from Hacking and
Churchill, Wyton, Huntingdon, Cambs., U.K., and were used 3 to 6 days after delivery.
Inoculation of mice. Groups of eight mice were inoculated intranasally under light ether anaesthesia with a 20 gl
vol. as described by Dimmock & Kennedy (1978). Treatment of mice with DI SFV comprised two inoculations of a
DI SFV preparation 2 h apart. The second inoculum contained 10 LDs0 (6000 p.f.u.) standard SFV. Possible
immunogenic stimulation by DI SFV was controlled by using non-infectious u.v.-irradiated standard SFV (UVSFV) of the same antigenic mass as determined by haemagglutination.
Tissue sampling. When tissue samples were required, mice were killed with ether. Blood was obtained from the
heart and serum was stored at - 70 °C. Spleen, olfactory lobes and brain (minus olfactory lobes) were dispersed in
Medium 199 containing 2~ newborn calf serum and stored at -70 °C.
U.v. irradiation. This was done as described by Dimmock & Kennedy (1978)except that the virus was placed 10
cm below a u.v. lamp (Gelman Sciences, Northampton, U.K.) for appropriate times. The dosage was 14gW/s/cm 2
and was checked on each time of usage with a Blak-Ray u.v. meter (Model J225, Ultraviolet Products Inc., San
Gabriel, Ca., U.S.A.).
Haemagglutination assay. These were performed according to Clarke & Casals (1958). Goose red blood cells
were obtained from CAMR, Porton Down, Wilts., U.K.
Inhibition o f S F V multiplication by D I virus
10
I
8
!
~
I
I
Y'-'-'--I
o
~
1911
--
6o
i
0
I
I
i
i
1
2
3
4
5
Time (days)
Fig. 1. Growth curves of SFV in CFLP mice after intranasal inoculation of 10 LDso. Groups of eight
mice were killed at various times post-infection and tissues assayed for infectivity as described in Methods. Error bars are + 1 S.E.M.On day 5 post-infection only one mouse was alive for sampling. O, Brain
(minus olfactory lobes); A, olfactory lobes; O, spleen; A , serum.
Interference assays. Two assays, the RNA synthesis inhibition assay (RSIA) (Barrett et al., 1981) and the yield
reduction assay (YRA), were used to quantify the interfering activity of DI virus. The latter is a modification of an
assay originally described by Bellett & Cooper (1959) and since modified by Kowal & Stollar (I 980). DI SFV is
diluted in a diluent of maintenance medium [GMEM plus 2 % newborn calf serum and 2 ~tg/ml actinomycin D (to
inhibit any induction of interferon)] containing standard virus at an m.o.i, of 5 p.f.u./cell. Samples (250 ~tl)of each
dilution are inoculated onto monolayers of 2 x 105 L929 cells in glass vials and left for 1 h at 37 °C. The inoculum is
removed and replaced by 1 ml maintenance medium and incubation continued at 37 °C. At 16 h post-infection incubation is stopped and cultures are assayed for infectivity. The number of defective interfering virus units (DIU)
in a DI SFV preparation is defined as the reciprocal of the dilution where standard virus production is reduced to
50% of controls inoculated with standard virus alone.
Histologicalprocedures. Mice were killed with ether and the brains and spinal cords removed intact. Part of the
brain was removed for infectivity assay and the remainder fixed in neutral buffered formalin. Tissues were prepared for paraffin embedding and sectioning by standard procedures. Sections were stained with haematoxylin
and eosin.
Immunochemical staining. Paraffin sections were reacted with convalescent antiserum prepared by infecting rabbits with the avirulent A774 strain of SFV and 3 weeks later with the ts ÷ strain. Positive antibody-antigen reaction
was located with biotin-avidin (Vectastain; Vector Laboratories, Burlingame, Ca., U.S.A.) or peroxidase-antiperoxidase (Dakopetts A/S, Guldborgvej, Copenhagen, Denmark) methodology. There was positive immunohistochemical staining with anti-SFV diluted up to 1/120.
RESULTS
In a previous study we h a v e shown that D I S F V p r e p a r a t i o n s p4 and p l 3 a p r o t e c t e d m i c e
against infection w i t h 10 LDs0 s t a n d a r d S F V by two a p p a r e n t l y different m e c h a n i s m s (Barrett
& D i m m o c k , 1984c). Properties o f the two D I virus p r e p a r a t i o n s and the ways in w h i c h they
m o d u l a t e d various p a r a m e t e r s in the m o u s e are s u m m a r i z e d in T a b l e 1. T h i s shows that alt h o u g h the D I S F V p r e p a r a t i o n s c o n t a i n the s a m e a m o u n t o f S F V a n t i g e n and p r e v e n t d e a t h o f
the s a m e p r o p o r t i o n of mice, a n i m a l s inoculated w i t h D I S F V p l 3a were resistant to challenge
w i t h 100 L D s o at 21 days post-infection, w h e r e a s D I S F V p4-treated m i c e were c o m p l e t e l y susceptible. T o investigate these differences we h a v e i n o c u l a t e d m i c e w i t h 10 LDs0 plus one or
o t h e r of the two D I S F V p r e p a r a t i o n s and d e t e r m i n e d the distribution o f infectious virus in a
n u m b e r o f tissues at intervals after infection.
Growth curve o f standard S F V in mice after intranasal inoculation
Fig. 1 shows the g r o w t h curves o f 10 L D s o s t a n d a r d S F V in C F L P m i c e after intranasal inoculation. T h e serum, spleen, b r a i n (minus olfactory lobes) a n d olfactory lobes were assayed for in-
4
pl3a
3 x l0 T
Infectivity
(p.f.u./ml)
I x 108
60
U.v. (s)*
100
,
50
6
Interference titre (DIU/ml)t
x
•
Before u.v.
After u.v.
56
8
59
Protection
(~)§
60
<9, <9, <9, 9, 10, 18, 22, 22, 141,
398, 501, 1000
5
81
Neutralizing antibody titre¶
<2, <5, <9, <9, < 2 0 , 2 , 9 , 3 2 , 2 0 0 0 ,
2000, 3162
challenge (7oo)11
Survivors of
&
)k
* U.v. irradiation time (in seconds) used to inactivate standard virus in the DI SFV preparation prior to use for protecting mice.
"~Protocol for RSIA is given in Methods.
:~ Data from Barrett & Dimmock (1984c).
§ Expressed as a percentage of mice inoculated with 10 LDso standard SFV plus DI SFV.
1[Protected mice in the previous column were challenged with 100 LDs0 at 21 to 24 days post-infection.
¶ Serum at day 13 post-infection, obtained by the plaque reduction assay. Mice inoculated with DI virus alone have no detectable antibody titre.
Antigenic
mass
(HAU/ml)
4
Passage
history
p4
Protection of mice by DI SFV:~
DI SFV preparation
T a b l e 1. Properties of the DI S F V preparations used to protect mice
t'~
©
,..]
m
>
,..]
Inhibition of S F V multiplication by DI virus
1913
fectivity. Virus was first seen in the olfactory lobes at 12 h and this increased up to 3 days post-infection. Virus appeared to spread posteriorly and was first detected in the rest of the brain at 1
day post-infection. Infectivity increased until 4 days post-infection when peak titres (up to 10 l°
p.f.u./brain) were detected. Virus was only detected in spleen and serum from day 2 post-infection where virus titres reached 106 p.f.u./mouse. Titres between mice were very consistent and
the calculated error was correspondingly small. Mice showed clinical signs of disease on day 3
and died either on day 4 or day 5 post-infection. It was rare for an animal to survive until day 6.
Multiplication of virus in mice co-infected with D I and standard S F V
Groups of eight mice were inoculated with DI SFV plus 10 LDso standard virus or 10 LDso
standard virus plus UV-SFV according to the protocol of Dimmock & Kennedy (1978). UVSFV [ the equivalent of 4 haemagglutinating units (HAU)/ml of SFV antigen] was used to control for any immunogenic activity of the DI virus (also 4 HAU/ml) and also showed that uptake
of infectious virus in the nose was not being impeded. Properties of the DI virus preparations
used are summarized in Table 1. Mice were killed at intervals after inoculation and the infectivity present in serum, spleen, brain (less olfactory lobes) and olfactory lobes determined. The results obtained for the two DI SFV preparations examined are shown in Fig. 2 and 3. All mice not
treated with DI virus died by day 5 post-infection; hence, on this day only reduced numbers were
available for sampling.
Mice treated with DI S F V p4
Comparison of the infectivity titres in mice treated with 10 LDs0 plus DI SFV p4 with those
found in mice inoculated with 10 LDs0 plus UV-SFV (Fig. 2) shows that the DI virus-treated
mice can be divided into two classes: (i) those which have iitres reduced by > 99 % and (ii) those
which have titres that were not significantly different. Overall 58 % of the infectivity titres from
the four tissues examined were in the former group. This figure compares favourably with lethality studies where 60% survived. Thus, we equate the first group with mice that would be protected from death by DI virus and the second group with mice that would die with the disease
following its normal course (Dimmock & Kennedy, 1978). Examination of Fig. 2 shows that up
to day 2 post-infection, infectivity titres in the brain, olfactory lobes and spleen were virtually
identical in DI-treated and untreated mice whereas infectivity titres in the serum were lower in
DI-treated mice than in untreated mice. However, the majority of samples from DI virus-treated
mice taken from day 3 onwards had lower infectivity titres than those of untreated mice. Many
of these samples, and all samples taken from day 7 post-infection onwards, had undetectable
levels of infectivity ( < 400 p.f.u./tissue).
Mice treated with DI S F V p13a
Infectivity titres obtained from mice infected with 10 LDso plus DI SFV pl3a can again be
divided into two classes (Fig. 3). Sixty-eight H of all the infectivity titres from the four tissues
examined were in the group defined as having titres reduced by > 99% compared with mice not
treated with DI SFV. This figure is close to the 59 % survival achieved by treatment with DI SFV
p 13a in lethality experiments. The pattern of infectivity titres in DI SFV p 13a-treated mice was
different from that found in DI SFV p4-treated mice. Whereas the latter group had infectivity
titres identical to untreated mice up to day 2 post-infection, mice that had been inoculated with
10 LDso plus DI SFV pl3a had reduced infectivity titres at all time points examined. There was
little virus in serum (compared with other tissues) of DI virus-treated mice, while infectivity
titres in the spleen on days 2 and 3 post-infection showed little reduction compared with mice
that did not receive DI virus. These results demonstrate that DI virus not only prevents the encephalitis but also prevents spread of virus in other tissues. Inspection of infectivity titres of
mice that comprise the group of individuals in which 'titres were reduced by > 99 %' showed that
the majority contained no detectable virus ( < 400 p.f.u./tissue). However, in other experiments,
a more sensitive assay has shown that many of these mice have levels of infectious virus ranging
from 10 to 100 p.f.u./mouse, indicating that the majority of mice were indeed infected; only 27 %
had less than 10 p.f.u./mouse on day 4 post-infection. At days 7, 11 and 14 post-infection virus
1914
A. D. T. B A R R E T T A N D O T H E R S
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,-
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[-.,
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¢¢3
t'-4
I
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o s n o t u / ' n ' j " d 0, ~ o I
Fig. 2. Multiplication of SFV in mice inoculated with 10 LDso and treated with DI SFV p4. Eight mice
were inoculated with 10 LDso plus UV-SFV or 10 LDso plus DI SFV and were sacrificed at various
times after infection. (UV-SFV is inactivated standard virus containing the same a m o u n t of antigen as
DI virus and is included to control for possible immunogenic effects.) Curves represent m e a n infectivity
titres of detectable virus of samples from mice inoculated with 10 LDso plus UV-SFV. Arrows with an
adjacent n u m b e r refer to the n u m b e r of mice with undetectable infectivity. O , 10 LDso plus UV-SFV;
A , 10 LD5o plus DI SFV.
Inhibition of SFV multiplication by DI virus
I
I
I
I
•
•
I
I
I
I
I
1915
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I
4"4F4
~-.~-i-~o~
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Fig. 3. Multiplication of SFV in mice inoculated with 10 LD5o and treated with DI SFV pl3a. Other
details are as described in the legend to Fig. 2.
1916
A.D.T.
BARRETT
AND
OTHERS
Table 2. Abnormal distribution of infectivity in tissues of some mice inoculated with 10 LDso plus
DI S F V pl3a
Infectivity (log~o p.f.u./tissue)
¢.
A
Daypost-in~ction
2
Mouseno.
1"
2
6
Brain
7.3
<2.0
<1.7
Ol~c~lobes
6.9
<1.7
5.7
3
1"
2
4
6
8
8-8
<1.3
<1.3
4.0
5.2
5
1"
2
3
14
4
.~
Spleen
3.2
2.6
3.0
Semm
4.4
<2-6
<2.6
6.5
<2.0
5.2
4.2
<1-7
4.0
3.6
3.0
3.3
<1.4
5.2
4.3
<2.0
3.1
<2.6
7.5
3.4
<1.6
5.7
5.1
<1.7
2.8
<2.0
2.7
4.6
4.1
3.3
4.9
<1.7
<2.0
<2.0
*Controlmicewereinoculated withl0LDs0plus UV-SFV. These werenotgiven Dlvirus.
was not detectable ( < 400 p. f. u./mouse) in any of the tissues with one exception at day 14 which
had a brain infectivity titre of 1049, but no virus in the other three tissues examined.
Disproportionate reduction in specific organ infectivity in infections modulated by DI S F V p l3a
As discussed above, mice inoculated with D I SFV p l 3 a fell into two classes: (i) those with
infectivity titres reduced by > 99% compared with mice infected with 10 LDs0 plus U V - S F V
and (ii) those with titres unaffected by inoculation o f DI virus. In the majority of mice, the levels
of infectivity in each of the four tissues investigated were either consistently low or consistently
high. The latter were indistinguishable from mice inoculated with SFV alone (Fig. 1). However,
nine mice treated with DI virus p l 3 a had titres which did not fit either pattern. These had high
titres in one or more tissues and low titres in the others (Table 2). F o r example, mouse 6 on day 2
had no detectable virus in brain and serum and yet there were high titres in the olfactory lobes
and spleen whereas the control mouse (mouse 1 on day 2) had similar titres in olfactory lobes and
spleen but > 105-fold more virus in the brain and > 10~8-fold more virus in the serum. It must be
emphasized that only 9/62 (14.5 %) had this unusual pattern of virus infection and that none of
these mice showed clinical signs of sickness. The effect was specific to D I virus p t 3a and none o f
the mice treated with D I SFV p4 showed an unusual pattern of virus infectivity. Particularly
noteworthy is the presence of virus in brain at 14 days after infection when one would have
expected immune processes to have cleared virus completely.
Failure to detect DI virus in DI virus-treated mice
It was of obvious interest to follow the levels of D I virus in the above experiments. In an
attempt to do so we used both the Y R A and the R S I A as these appear to measure different
parameters of interference (Barrett et al., 1984). Both assays failed to detect D I virus in either
brain or olfactory lobes of D I virus p4- or p 13a-treated mice. This result was unexpected since we
have demonstrated elsewhere that inoculation of D I virus prevents death ( D i m m o c k &
Kennedy, 1978; Barrett & Dimmock, 1984b, c) and have described above how D ! SFV inhibits
virus multiplication. Possible explanations are that the assays m a y not be sufficiently sensitive
(the R S I A detects > 10515 D I particles/250 txl: Barrett et al., 1981), that D I virus which is registered by these assays does not interfere in mice or that DI virus is not p r o p a g a t e d in the mouse.
Absence of histopathology and virus antigen in brains of mice treated with DI S F V
Brains from mice treated with either DI SFV p4 or p l 3a were obtained on days 4 and 11 postinfection and stained sections examined for histopathological changes. Mice infected with 10
Inhibition of S F V multiplication by D I virus
1917
Table 3. Immunity generated & mice inoculated with graded doses of virulent and avirulent S F V
Virus
Virulent
Avirulent
Dose
inoculated
(loglo p.f.u.)*
2.8
1-8
0.8
Titre in brain at day 4
(log10 p.f.u./mouse)
< 1~6, 8.4, 8.3, 8.9
All < 1.6
All < 1.6
1.8
All < 1.6
6-6
5-6
4.6
3-6
2.6
7-1, 6-9, 6.5, 5.9
6.5, 6.2, 6-2, 6-2
6-4, 8.2, 6-2, 6-4
6.4, 6.2, 1.3, 6-6
3.0, < 1-3, < 1.3, < 1.3
1.6
All < 1.3
None§
Survivors of
primary infection
r ~ ' ~ ' ~ r
t
%
2/5
40
5/5
100
5/5
100
5/5
100
Survivors of
challenge
"~
•
:~
1/2
50
1/5
20
0/5
0
0/5
0
4/5
3/5
3/5
5/5
5/5
5/5
80
60
60
100
100
100
4/4
3/3
3/3
4/5
1/5
0/5
100
100
100
80
20
5/5
100
0/5
0
0
* Virus was inoculated intranasally in a vol. of 20 pl as described in Methods. 102.8p.f.u, virulent SFV is 1 LD50.
Virulent SFV is the standard virus used throughout.
t Number of mice surviving/number of mice inoculated.
:~Number of mice surviving/number of mice inoculated. Challenged with 100 LDso.
§ Mock-infected with medium only.
LD50 + UV-SFV showed extensive neuronolysis throughout the brain including the olfactory
bulbs, occasional but pronounced focal degeneration notably in the pyramidal cell layer of the
hippocampus and mild perivascular cuffing as described previously (Crouch et al., 1982). However, brains from DI virus-treated mice were indistinguishable from mock-inoculated controls
on both days tested, The presence of virus antigen in brains of infected mice not treated with DI
virus could be readily demonstrated by immunochemical staining and was sometimes
coincident with pathological lesions. However, some apparently healthy neurons also contained
antigen, for example in the pyramidal cell layer of the hippocampus and in the cerebral cortex.
Positive immunohistochemical staining of virus antigen was also found in the cytoplasm of
otherwise healthy neurons and glial cells, particularly in thalamic nuclei, substantia nigra and
brain stem. Cells in spinal cord and Purkinje cells in cerebellum were also sometimes positive.
Conversely, in some anatomical areas there were sites of histopathological change such as cellular degeneration and perivascular cellular reaction which contained no demonstrable antigen.
Similarly, olfactory lobes did not show marked concentrations of antigen by immunohistochemistry. N o virus antigen was detected in brain or spinal cord of DI virus-treated mice.
DISCUSSION
In this study we have shown that the two DI SFV preparations examined, p4 and pl3a, both
inhibit the multiplication of standard virus in the mouse. Inoculation of mice with 10 LDso plus an
amount of UV-SFV equivalent to the DI virus showed that modulation was not due to blocking
of cellular receptors or to the immunogenicity of DI SFV. Mice treated with DI SFV pl 3a had
low infectivity levels throughout infection, while DI SFV p4-treated mice had infectivity titres
identical to untreated mice for the first 2 days of infection (Table 4). Titres in p4-treated mice
then declined and from day 3 onwards were similar to those in p 13a-treated mice. In both experimental groups virus was cleared by day 7 with one exception which had a high level of brain
infectivity at day 14 post-infection. Thus, we have the situation in DI virus-treated mice that
standard virus is no longer virulent and multiplication of standard SFV is contained by some
unknown mechanism as a subclinical infection. This is all the more remarkable as Table 3
demonstrates that, in the absence of DI virus, any mouse that is infected develops a lethal encephalitis (i.e. ID50 = LDs0).
The infectivity data reported above present a paradox since infected mice treated with DI
SFV p4 have the same distribution and amount of infectivity at day 2 post-infection as untreated
mice and we therefore would expect there to have been a sufficient immunogenic stimulus to
1918
A. D . T. B A R R E T T A N D O T H E R S
Table 4. Percentage o f mice with 'normal" amounts o f infectivity* in tissues at 2 days postinfection
Inoculum
10 LDso + DI SFV p4
10 LDso ÷ DI SFV pl3a
Olfactory lobes
100
50
Brain
100
25
Spleen
100
75
Serum
100
0
* Defined as having > 1~ of the mean infectivity titre of control mice infected with 10 LDso plus UV-SFV
(eight mice per group). Data were taken from Fig. 2.
generate a protective immunity. However, these mice were unable to resist challenge by 100
LDso SFV at 3 weeks after infection and the disease followed its usual lethal course (Barrett &
Dimmock, 1984c). Conversely, as mice treated with DI virus pl 3a had much lower levels of infectivity, one might have expected immunity to be impaired but such mice resisted challenge
completely.
Levels of neutralizing antibody in both sets of mice were similar and less than in mice that had
experienced infection with avirulent SFV; the distribution of titres in mice treated with DI virus
p4 or pl3a were also very similar (Barrett & Dimmock, 1984c; Table 1). One possible explanation is that infected mice treated with DI SFV pl 3a synthesize an immunizing amount of noninfectious SFV antigen but this appears unlikely as immunocytochemistry failed to show the
presence of SFV antigen in sections of brain and there was no sign of immune cell infiltration.
The failure of mice to establish protective immunity may therefore be due to an immunosuppression peculiar to DI virus p4, although this is unlikely to be general immunosuppression.
We know of no precedent for this observation.
In an attempt to resolve the problem outlined above, mice were infected intranasally with different quantities of virulent or avirulent SFV to determine whether, in the presence of low or
undetectable levels of SFV, mice could develop immunity to challenge (Table 3). Firstly, the results show that for virulent SFV the LDs0 is also the IDs0 and mice either develop high titres of
virus in brain or none at all. This contrasts with the intermediate levels of infectivity seen above
in infections involving DI virus. Secondly, mice inoculated with less than 1 LDs0 had no detectable virus in brain at day 4 post-infection and were susceptible to subsequent challenge with 100
LD50. (An odd survivor is also seen on occasion in mice with no previous experience of SFV.)
Inoculation of mice with > 1 LD50 equivalent of avirulent SFV (i.e. > 103.6 p.f.u.) resulted in
virus titres in brain of over 106 p.f.u, on day 4 post-infection (the peak of infectivity; A. D. T.
Barrett & N. J. Dimmock, unpublished observations) and these survived challenge with 100
LDs0. The majority of mice inoculated with 1016 or 102.6p.f.u, did not have virus in brain at day
4 and did not survive challenge. We therefore conclude that, in an SFV infection where DI virus
has not been inoculated, mice that have no detectable virus in brain develop no protective
immunity and will succumb to challenge. It is self evident that mice treated with DI SFV pl 3a
together with 10 LDs0 and which subsequently resist challenge by 100 LDso must express sufficient SFV antigen to stimulate protective immunity; however, the nature and distribution of this
SFV antigen is unknown at present.
Despite clear evidence that administration of DI SFV prevents disease and death (Barrett &
Dimmock, 1984b) and inhibits standard virus multiplication, we were unable to detect DI virus
in brain or olfactory lobes. It may be that the concentration of DI virus is low and cannot be
detected by the assays that register > 105.75DI particles/ml. Indeed, in a previous study an amplification step did reveal the presence of DI virus in mouse brain (Dimmock & Kennedy, 1978).
A similar problem of low levels of DI VSV after inoculation into adult mice was ascribed to the
age of the host (Holland & Villareal, 1975) as DI virus could be propagated and even generated
in the brains of newborn animals. Alternatively, the DI virus which interferes in vivo may be
different from the DI virus active in tissue culture and undetectable in assays in vitro. The latter
seems unlikely but since DI SFV R N A is heterogeneous (K/i/iri/iinen et al., 1981) the possibility
cannot be excluded.
Analysis of the infectivity titres in DI SFV p 13a-treated mice revealed that 14-5~ showed an
anomalous distribution of infectivity whereas distribution of infectivity in mice treated with DI
Inhibition of S F V multiplication by DI virus
1919
SFV p4 was always indistinguishable from the untreated control. In these p 13a-treated mice one
or more tissues contained a disproportionate amount of virus, as if D I virus was failing to inhibit
multiplication in that particular tissue. These results indicate that as well as controlling the level
of virus multiplication in infected tissues, DI SFV can cause a more subtle, possibly tissuespecific, modulation of infection. Furthermore, as the mice referred to above, including the individual with brain infectivity at day 14, were clinically normal, it is interesting to speculate that
they may have Survived carrying a persistent infection which has the potential to produce disease in later life.
In the above discussion we have dealt with the ways in which two D I SFV preparations differ
from each other without commenting on the possible basis of this difference. However, others
have found that SFV DI preparations produced in B H K cells in a similar way to p4 and 13a were
physically heterogeneous and had sequences that differed as a result of deletions, sequence
rearrangements and repetitions of parts of the standard virus genome (K/i~iri/iinen et al., 1981 ;
Pettersson, 1981 ; S6derlund et al., 1981 ; Lehtovaara et al., 1981, 1982). We therefore suggest
that it is possible that the two DI preparations p4 and pl 3a, which we find differ in their interference properties, do so because of differences in the sequence of their RNAs.
A.D.T.B. was supported by an S.E.R.C. studentship and this project was funded in part by the Medical Research Council. Actinomycin D was the kind gift of Merck, Sharp and Dohme.
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(Received 16 May 1984)