J. gen. Virol. (I98o),49, 215-22o
2I 5
Printed in Great Britain
Requirements for DI Particle Prophylaxis Against Vesicular
Stomatitis Virus Infection in vivo
(Accepted I8 January I98O)
SUMMARY
In contrast to biologically active DI particles, neither u.v.-inactivated standard
virus nor either of two different homologous u.v.-inactivated DI particles showed
any prophylactic effect when injected intracerebrally into mice concomitantly
challenged with VSV. Although u.v.-inactivated DI particles did not prevent
death when given with the challenge virus, they did significantly lengthen the time
until death occurred. Also, both u.v.-inactivated standard virus and DI particles
protected mice against late challenge (at 3 or lO days after treatment). Dosage
titrations of preparations of two different active DI particles showed significant
prophylaxis against simultaneous challenge with numbers of DI particles IO- to
Ioo-fold lower than those which gave no prophylaxis when u.v.-inactivated. Thus,
prophylaxis in this system required biologically active DI particles.
The suggestion made by Huang & Baltimore (197o) that defective interfering (DI) virus
particles may affect the course of virus disease has been tested by experiments with reovirus
(Spandidos & Graham, 1976), lymphocytic choriomeningitis virus (LCMV; Popescu &
Lehmann-Grube, I977), Semliki Forest virus (SFV: Dimmock & Kennedy, 1978) and
vesicular stomatitis virus (VSV, Holland & Villarreal, 1975). These studies suggested the
possibility that the presence of DI particles might be able to prevent or alter virus disease,
as had the very earliest work with 'incomplete virus' of influenza (yon Magnus, 195 I, 1954)
and of Rift Valley fever virus (Mims, 1956).
A critical question, however, is whether these DI particles are acting by specific interference with replication of the standard virus, or by another means. Doyle & Holland (1973)
showed in mice that large numbers of highly purified VSV DI particles gave prophylaxis
against concomitant intercerebral VSV challenge. No protection was seen against heterologous viral challenge, indicating that interferon was not involved. However, since the
injected DI particles were in sufficient number (5 × lO1° particles) to provide a sizeable
antigenic stimulus in these mice, an immune response was to be expected, and in fact it was
shown that by IO days after injection of purified VSV DI particles alone at these levels,
mice were immune to challenge by standard virus. Crick & Brown (1977) further demonstrated that inactivated VSV DI particles could provide protection against late challenge
within 2 days. Also, some slight non-specific protection against heterologous virus challenge
was observed at 2 days. Furthermore, a slight protection against concomitant homologous
VSV challenge was seen by these workers and this indicated that interference was not the
only effect exerted by the DI particles.
To determine to what extent true homologous interference by DI particles with the
replication of challenge virus contributed to the prophylaxis seen by Doyle & Holland
(I 973), we repeated the original studies. We compared the degree of intracerebral protection
provided by large antigenic amounts of u.v.-inactivated DI particles with that provided by
u.v.-inactivated standard virus. We also carried out quantitative dosage-protection titrations
for two different active DI particles.
Complete u.v.-inactivation of standard virus and of DI particles was carried out by
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Short communications
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lO s
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Fig. T. Failure of u.v.-inactivated standard virus or of u.v.-inactivated DI particles to protect mice
from intracerebral challenge. The number of p.fiu, of virus isolated from the brains of mice at the
time of death due to virus infection is plotted against the day of death (the day of inoculation is
day o). The brains were homogenized in Io ml medium and titrated on BHK 2~ cells. Mice were
injected intracerebrally with: (a) 5 x ~ol° u.v.-inactivated standard virus, (b) 7 x io n u.v.-inactivated
MS DI particles, or (c) Io n u.v.-inactivated CAR 4 short DI particles and concomitantly challenged
with loo p.f.u, of standard virus. Note the difference in scale between panel (a) and the other two
panels. The open circles in all panels show the results obtained with IOO p.f.u, of standard virus
alone and the closed circles the results with ioo p.f.u, standard virus mixed with u.v.-inactivated
virus or DI particles. Samples of the same DI particle pools were injected without u.v.-inactivation
and these provided significant protection (shown in Table ]).
e x p o s u r e for 4 ° rain at a distance o f 6 c m f r o m a Sylvania G 8 T 5 germicidal lamp. N o
infectivity r e m a i n e d in the s t a n d a r d virus (over IO12-fold inactivation), n o r were similarly
exposed D I p a r t i c l e p o o l s a b l e to replicate or to interfere. All o t h e r p r o c e d u r e s are as
described in D o y l e & H o l l a n d (i973). Samples o f each virus o r D I p a r t i c l e p o o l were irradia t e d o r left u n t r e a t e d a n d all were stored frozen at - 7 o °C with only one freeze-thaw for
each sample. M i c e were y o u n g adults m a t c h e d for age f r o m a single b r e e d e r b u t were n o t
m a t c h e d litter mates.
Previously, D o y l e & H o l l a n d (I973) showed t h a t i n t r a c e r e b r a l injection o f mice with
a p p r o x . IOO p.f.u, o f VSV I n d i a n a serotype is fatal within 2 to 3 days, while c o n c o m i t a n t
injection o f 5 x i o 1° M S D I particles (isolated f r o m a stock o f the M u d d - S u m m e r s strain o f
VSV used in this l a b o r a t o r y ) gave nearly c o m p l e t e p r o t e c t i o n (I I o u t o f I2 survived). T o
d e t e r m i n e if this D I - m e d i a t e d p r o p h y l a x i s was due to a n i m m u n e response, 5 x i o TM u.v.inactivated s t a n d a r d virus particles were injected c o n c o m i t a n t l y with too p.f.u, o f live
s t a n d a r d virus. This u.v. i n a c t i v a t i o n o f VSV does n o t alter virus p r o t e i n antigenicity as will
be shown below. I n fact, the m a j o r target is the R N A genome, with one-hit kinetics linearly
d e p e n d e n t u p o n the length o f s t a n d a r d virus o r o f D I particle g e n o m e R N A (F. M.
H o r o d y s k i & J. J. H o l l a n d , u n p u b l i s h e d data). Eleven o f the T2 mice injected with the
a b o v e m i x t u r e died a n d there was no significant difference in the average d a y o f d e a t h
c o m p a r e d with c o n t r o l s injected with IOO p.f.u, s t a n d a r d virus a l o n e (Fig. I a). N o r was
there a significant r e d u c t i o n in p . f . u . / b r a i n as c o m p a r e d to c o n t r o l s in Fig. I (a).
Since u . v . - i r r a d i a t i o n o f D I particles destroys their a b i l i t y to replicate a n d to interfere,
we next u . v . - i r r a d i a t e d p r e p a r a t i o n s o f two different D I particles to d e t e r m i n e w h e t h e r
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217
T a b l e I. Effect of standard virus or D I particles upon
intracerebral challenge by VSV*
Prophylactic treatmeut
Expt. 1 (u.v.-standard virus)
None
5 X IO 1° u . v . - s t a n d a r d
Expt. 2 (u.v.-DI particles)
None
7 x io TM u.v.-MS DI
None
1011 u.v.-CAR 4 S DI
Expt. 3 (late challenge)
None
5 x IOTM u.v.-standard
5 x lOTM u.v.-standard
None
Day of
challenge
No. of
survivors
Average day
of death
IOO p.f.u,
ioo
o
0/8
o
1/12
4"8~2'5
4.i±i.i
lOO
IOO
o
o
o/8
0/8
I OO
0
O
I/ II
I/II
Challenge
IOO
IOO
IOO
3 x lO8
o
3
IO
o/8
5/9
3/7
100
O
IOO
1oo
3
IO
8/8
7/8
IOO
127
o
o
0/8
IO/II~
5 X 10 TM M S D I
IOO
0
3/8§
5 x lO9 MS DI
5 x IOs MS DI
None
3x lO1° CAR 4 S DI
3x lO9 CAR 4 S DI
3 x 10s CAR 4 S DI
3 X 107 CAR 4 S DI
3 x io 6 CAR 4 S DI
3 x lO5 CAR 4 S DI
IOO
IOO
IOO
IOO
lOO
IO0
lOO
ioo
lOO
o
o
o
o
o
O
o
o
o
6/8
I/8
o/8
IO/II
6/8
4/8
2/8
2/8
0/8
Io 11 u.v.-CAR 4 S DI
I011 u.v.-CAR 4 S DI
Expt. 4 (DI dosage curve)
None
5 x IO1° MS DI
I/ II
2"8+0' 5
3"8±1"O
2"8±0"4
5"8±2"5
4"8+2"5
6"8
I4
2-8+0'4
(t4)'{"
2.8+0.5
(I2)
7"2
(8)
6"4
2"8 xo-5
(6)
(8)
9"8
5"7
6"0
3"0
* All of these experiments (and those in Fig. 1) were done with a single pool of each virus or DI particle
(samples of which were treated with u.v. or left untreated as indicated and tested after storage at - 7o °C
with only one freeze-thaw cycle for each tested sample).
t Day of death is in parentheses when only 1 or 2 died.
From Doyle & Holland, 1973.
§ In this experiment the ambient temperature was 2o to 2I °C while the other experiments were done at
25 to 26 °C ambient temperature. There were fewer survivors under the stress of the lower temperature.
p r o p h y l a x i s m i g h t be d u e to t h e m e r e p h y s i c a l p r e s e n c e o f D I p a r t i c l e s w h i c h w e r e replic a t i v e l y i n a c t i v e a n d n o n - i n t e r f e r i n g . I n t w o d i f f e r e n t e x p e r i m e n t s 7 × IO TM u . v . - i n a c t i v a t e d
M S D I p a r t i c l e s (Fig. I b) o r lO 11 u . v . - i n a c t i v a t e d C A R 4 s h o r t D I p a r t i c l e s (Fig. I e) w e r e
e a c h i n j e c t e d i n t o m i c e c o n c o m i t a n t l y w i t h IOO p.f.u, o f s t a n d a r d virus. T h e C A R 4 s h o r t
D I p a r t i c l e w a s i s o l a t e d f r o m t h e B H K 2 I VSVI~D C A R 4 line o f p e r s i s t e n t l y i n f e c t e d cells
a f t e r 2 years o f p e r s i s t e n t i n f e c t i o n ( H o l l a n d et al. I976). T a b l e 1 s h o w s t h a t t h e r e w a s n o
d i f f e r e n c e in t h e n u m b e r s o f m i c e s u r v i v i n g as c o m p a r e d to c o n t r o l s ; h o w e v e r , an i n t e r e s t i n g
d i f f e r e n c e in t h e a v e r a g e d a y o f d e a t h w a s o b s e r v e d w i t h b o t h D I particles. A s s h o w n in
Fig. I (b a n d c), this effect w a s slight f o r t h e M S D I p a r t i c l e s b u t w a s s t r i k i n g w i t h t h e C A R 4
s h o r t D ! particles. N o t e also t h a t t h e yields o f p . f . u . / b r a i n a r e s o m e w h a t r e d u c e d b y t h e
C A R 4 s h o r t D I particles.
F r o m t h e a b o v e results, w e c o n c l u d e t h a t t h e p r o p h y l a c t i c effect o f V S V D I p a r t i c l e s in
m i c e c h a l l e n g e d w i t h V S V is n o t d u e to t h e i r i m m u n e r e s p o n s e , since n e i t h e r i n a c t i v a t e d
s t a n d a r d virus n o r i n a c t i v e D I p a r t i c l e s c a n p r o v i d e this effect. T o c o n f i r m t h a t t h e u . v . i n a c t i v a t e d s t a n d a r d virus a n d D I p a r t i c l e s e m p l o y e d a b o v e w e r e a n t i g e n i c , w e i n j e c t e d
m i c e i n t r a c e r e b r a l l y w i t h 5 × IO1° u . v . - i n a c t i v a t e d s t a n d a r d virus o r IO11 u . v . - i n a c t i v a t e d
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Short communications
CAR 4 short DI particles before challenging them with standard virus at 3 or IO days after
injection. As seen in Table I, we observed significant protection at 3 and at Io days p.i. of
either inactive standard virus or of inactive DI particles.
It was important to discover whether the massive doses of active DI particles which had
been given were necessary for prophylaxis. Dosage titrations were done with preparations
of both the MS DI particle and the CAR 4 short DI particle with a challenge dose of
IOO p.f.u./mouse. In Table I it is shown that we could protect mice with Ioo-fold lower doses
of active CAR 4 short FDI particles than were used previously. However, relatively large
numbers of DI particles were necessary to provide protection against intracerebral challenge.
Interestingly, to-fold higher dosages of the MS DI particles than of CAR 4 short DI
particles were required to provide significant protection against challenge. It should be
noted that these smaller numbers of active DI particles provided significant prophylactic
protection and that this was not provided by lO- to Ioo-fold higher levels of u.v.-inactivated
virus or DI particles. This is strong evidence that the biological activity of DI particles is
required and not mere antigenic mass.
Our results also show that the antigenicity of these VSV DI particles is sufficient to
protect mice from challenge within days after injection. This agrees with an early report
(Doyle & Holland, 1973) that DI are immunogenic when given IO days prior to challenge,
and with the more unexpected finding of Crick & Brown (t977) that they also protect
against challenge within as little as several days. Crick & Brown (I977, I978) also observed
a sparing effect of inactivated DI particles against concomitant challenge which we have
not observed here. This difference between their results and ours is probably due to their
use of serial dilutions of test and control virus. Also, they observed a stronger effect when
the intramuscular route of inoculation was used and we have restricted the present study to
intracerebral inoculation.
In our data there are two interesting indications of another effect of injecting large
numbers of DI particles in addition to homologous virus interference and antigenic stimulation. Firstly, there is the prolongation of the course of disease seen with the u.v.-activated
DI particles (especially the CAR 4 short DI), even though they provided no protection
against death. This confirms the report of Crick & Brown (1977) that DI particles can exert
other (probably non-specific) protective effects in addition to true homologous autointerference. We did not observe this prolongation of life with antigenically equivalent
amounts of u.v.-inactivated standard virus. Secondly, the minimum number of the CAR 4
short DI particles needed to protect mice against challenge was less than that required for
protection by MS DI particles. Differences in the ability of different DI particles and of
standard virus to induce interferon may be involved. Certainly work by Marcus & Sekellick
(1977) indicated that there are such differences. They found that a DI particle which contains
'snapback R N A ' (RNA which is completely self-complementary) induced high levels of
interferon, while another DI particle with no snapback RNA was a poor interferon inducer.
Perrault & Leavitt 0977) showed that while the CAR 4 short DI particle RNA was completely self-complementary, the MS DI particle showed only a limited amount of selfcomplementarity, and so may be a less efficient interferon inducer. Preliminary studies
have shown that 3 x lO1° CAR 4 DI particles induced small amounts of interferon in the
brains of injected mice, but equivalent numbers of MS DI particles induced no detectable
interferon. However, it should be noted that Frey et al. (1979) found no correlation between
snapback RNA content of VSV DI particles and their ability to induce interferon, so
structure may not be relevant.
As has also been seen with SFV (Dimmock & Kennedy, I978) and reovirus (Spandidos &
Graham, I976), rather large numbers of DI particles are necessary for prophylaxis, For
example, Dimmock & Kennedy (1978) reported a requirement for 4 × Io 5 'p.f.u. equiDownloaded from www.microbiologyresearch.org by
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219
valents' of DI particles for intranasal protection. Since in these systems the lethal dose of
standard virus can be produced by the yield from one infected cell, DI particles probably
must co-infect every cell initially infected by standard virus for effective prophylaxis. This
is the most likely reason why large numbers of DI particles are required, but other explanations are possible.
The above results, in agreement with those of Dimmock & Kennedy 0978), indicate that
active, replicating DI particles are required for efficient prophylaxis. In addition, we find
evidence for other less striking forms of DI protection, possibly related to the varying
abilities of different classes of DI particles to induce interferon, in agreement with Crick &
Brown (~977, I978). Finally, Faulkner et al. (I979) have shown very recently that VSV DI
particles cause strong homologous auto-interference and prevent neuron death for many
days in dissociated neuron cultures from mice. These results and the present results strongly
suggest that true DI interference does take place in vivo.
We thank E. Bussey for excellent technical assistance. This work was supported by
U.S.P.H.S. Research Grant no. Ar 14627 to J . J . H . C . L . J . was supported by U.S.P.H.S.
Predoctoral Traineeship no. CA o5274 .
CHARLOTTE L. JONES*
Department of Biology C-oz6
University of California
San Diego, La Jolla
California 92093, U.S.A.
JOHN J. HOLLAND
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* Present address: Department of Cellular, Viral and Molecular Biology, College of Medicine, University
of Utah, Salt Lake City, Utah 84132, U.S.A.
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SPANDIDOS, D. A. & GRAHAM, A. F.
(Received 6 November I 9 7 9 )
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