bis virus, persistent infection may be readily established in mosquito cells but this ... Since a large number of Sindbis virus ts mutants is available (Atkins et al.
J. gen. Virol. (I979), 45, 2oi-2o7
2OI
Printed in Great Britain
Establishment of Persistent Infection in BHK-21 Cells by Temperature-sensitive Mutants of Sindbis Virus By G. J. A T K I N S Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland (Accepted 8 May I979) SUMMARY
Twelve temperature-sensitive (ts) mutants of Sindbis were examined for their ability to establish persistent infection in BHK-2I cells at 39 °C. Five of these mutants were able to initiate colony formation in infected cultures, which followed an extensive c.p.e. Two of the mutants were able to establish persistent infections which survived beyond the fifth cell passage p.i. The ability to initiate colony formation was correlated with low reversion of the ts mutation, or with ability to interfere with the multiplication of the wild-type virus. Virus released from persistently infected cultures was not temperature-sensitive. The restriction of virus multiplication in persistently infected cells operated prior to virus-specified RNA synthesis. It is concluded that in this system establishment of persistent infection depends on an inhibition of virus multiplication early in infection and occurs in only a small proportion of infected cells. INTRODUCTION
A number of RNA viruses are able to establish persistent infection in cell culture (Preble & Youngner, I975; Rima & Martin, 1976; Holland & Levine, I978). Several mechanisms have been proposed to explain this persistence; these include the generation of defective interfering (DI) particles, the action of interferon and the generation of ts mutants. For Sindbis virus, persistent infection may be readily established in mosquito cells but this virus normally causes lytic infections in mammalian cells. In mosquito cells both DI particles (Eaton, 1977) and ts mutants (Shenk et ak I974) are produced by persistently infected cultures. In addition, there have been two reports of persistent infection of mammalian cells by Sindbis virus. Schw6bel & Ahl 0972) described the establishment of persistent infection in BHK-2I cells by Sindbis virus. However, no information was obtained as to the mechanisms involved. Inglot et al. (I973) described a persistent infection of mouse L cells which was probably mediated by interferon. Since a large number of Sindbis virus ts mutants is available (Atkins et al. r974), and these mutants have been partially characterized (Atkins et al. I974; Brzeski et al. 1978; Brzeski & Kennedy, I978), the aim of the present work was to determine the extent to which these mutants could establish persistent infections in BHK-2I cells. This study is an extension of a previous one (Atkins, I976, t977) in which it was shown that RNA- mutants are defective in cytopathogenicity and involved studying the cells over a longer period following infection. METHODS
Cells. BHK-2ICI3 cells (Macpherson & Stoker, 1962) were grown in Glasgow-modified MEM supplemented with lO9/o newborn calf serum, IO~o tryptose phosphate broth, IOO units/ml of penicillin, IOO/~g/ml of streptomycin and 0"25 #g/ml of amphotericin B. Uninfected cells were split I/5 every 3 to 4 days. oo22-I317/79/oooo-3667 $ o 2 . o o ~ 1979 SGM
202
G.J.
ATKINS
Table I, Properties o f ts mutants used in present study Mutant Flo4 F294 H98 N2 N7 A82 AI2O F36 A93 HI8 N32 FI27 Wild-type
RNA phenotype* ----
--+ + + + + + ~-
Lesion Unknown pl 5or Unknown
Reference Unpublished results
E.o.p. (39/30 °C) of stock 3.8 × Io-5 < 10-5
p2I 5
Brzeski & Kennedy 0978)
I '5 )< 10-a 1"3 × 10 -4
p215 pI50, P144 PI44:~ p144 P144 Maturation§ Maturation Maturation
Unpublished results Unpublished results Brzeski et at. (1978) Brzeski et al. (I978) Brzeski et al. (1978) Unpublished results Unpublished results Unpublished results
2'9 × Io-4 5.8 × 1o 4 3'3 × IO-4 4"2 × lO-4 < IO-5 2'3 × l o - 4 I '9 × I o - 4 4"3 × 2o-3 I'I
* As determined by Atkins et ak (I974). t Accumulate a protein precursor of mol. wt. IS0000 on shift from 30 to 39 °C. :~ Accumulate a protein precursor of tool. wt. 144ooo at 39 °C. § Make nucleocapsids but not infectious virus particles at 39 °C.
Virus. All ts m u t a n t s are derived from the A R 3 3 9 strain o f Sindbis virus ( A t k i n s et al. 1974). To minimize the presence o f D ! particles, all stocks were g r o w n f r o m single plaques picked from m o n o l a y e r s i n c u b a t e d at 3o °C and were frozen at - - 7 ° °C in aliquots. Since the p r o p o r t i o n o f revertants in stocks p r e p a r e d in this way varies greatly, the same stocks o f virus were used in all the experiments described here; their revertant frequencies are shown in T a b l e ~. These were estimated by m e a s u r i n g the e.o.p, o f m u t a n t stocks at 3o °C and 39 °C in a plaque assay. Plaque assays were p e r f o r m e d using an overlay c o n t a i n i n g o'9 % a g a r and were incubated for 2 days at 39 °C or 3 days at 3o °C before plaques were scored. Establishment o f persistently infected cultures. B H K - / I cells were seeded into 15 c m 2 plastic flasks at a c o n c e n t r a t i o n o f a × io 5 cells/ml in 5 ml m e d i u m . The following day, when the cells were still subconfluent, virus was a d d e d to give an i n p u t multiplicity o f I p.f.u./cell in I ml o f m e d i u m . A f t e r allowing I h for a d s o r p t i o n , 4 ml o f g r o w t h m e d i u m were a d d e d . This m e d i u m was changed every 3 to 4 days for the d u r a t i o n o f the culture. Measurement o f virus yieM. BHK-21 cells were seeded at a c o n c e n t r a t i o n o f 2 × lO 5 cells/ml in 4 ml o f growth m e d i u m in 6o m m diam. plastic dishes. The following day, the m o n o l a y e r s were d r a i n e d a n d infected with o. 5 ml o f virus diluted to the a p p r o p r i a t e conc e n t r a t i o n in growth m e d i u m . After a d s o r p t i o n for 1 h at 39 °C, the virus i n o c u l u m was removed, 4 ml w a r m (39 °C) g r o w t h m e d i u m a d d e d to each dish and the cells i n c u b a t e d at 39 °C for the a p p r o p r i a t e time. The m o n o l a y e r s were then drained, washed with 4 ml w a r m (39 °C) phosphate-buffered saline (PBS), and 4 ml of w a r m growth m e d i u m a d d e d to each dish. The cells were incubated at 39 °C for the a p p r o p r i a t e time before the virus yield was harvested a n d frozen at - - 7 o °C for plaque assay. The degree o f interference in mixed infections was expressed as an interference ratio, defined as the yield o f wild-type (or s t a n d a r d ) virus a l o n e divided by the yield o f wild-type virus under c o n d i t i o n s o f putative interference. Virus-specified R N A synthesis. Cells were seeded into 6o m m diam. plastic dishes at a c o n c e n t r a t i o n o f 2 × IOs cells/ml in 4 ml m e d i u m . The following d a y triplicate m o n o l a y e r s were infected at the required multiplicity by d r a i n i n g the m o n o l a y e r s , a d d i n g o. 5 ml o f growth m e d i u m c o n t a i n i n g the virus a n d a d s o r b i n g for I h at 39 °C. The virus i n o c u l u m was then r e m o v e d a n d 4 ml o f growth m e d i u m c o n t a i n i n g 5/zg/ml a c t i n o m y c i n D ( w a r m e d to
Ts mutants o f Sindbis virus Table
2.
203
Ability of ts mutants to establish persistent infection at 39 °C
Mutant F294 H98 FlO4, A82, N7, N2 Hl8 N32 F127 AI2O, F36, A93, wild-type
No. of cultures No. of cultures showing colony surviving beyond R N A phenotype formation* passage 5* ---+ + + +
I 4 o 5 2 2 o
o 2 o 5 0 o o
* A total of five monolayers were infected with each mutant at an input multiplicity of I p.f.u./cell.
39 °C) added to the monolayers. Three h later, the monolayers were drained and 2 ml of growth medium containing 5/zg/ml actinomycin D and I #Ci/ml 3H-uridine (Radiochemical Centre, Amersham, Bucks, U.K. ; 47 Ci/mmol) added for a further 2 h. The monolayers were again drained, washed twice with 4 ml of PBS and dissolved in 2 ml of I ~o SDS solution. This solution was removed from the plate and mixed with 2 ml of ice-cold I o ~ trichloroacetic acid (TCA) solution. The solution was then left for 30 min on ice to allow precipitation to take place. The precipitates were collected on glass fibre discs by vacuum filtration, washed twice with 5 ~ TCA, twice with ethanol, then dried at 37 °C overnight and counted. RESULTS
Ability of ts mutants to establish persistent infection A total of 12 mutants were tested for their ability to establish persistent infection in BHK-2I cells at 39 °C (Table 2). Six of these mutants were R N A + (able to make virusspecified R N A at 39 °C), the other six R N A (unable to make virus-specified R N A at 39 °C). Other known properties of the mutants are summarized in Table I. Five infected cultures were set up for each mutant and the wild-type to test ability to establish persistent infection. Cells infected with any of the mutants or the wild-type eventually showed extensive c.p.e. If the medium was changed every 3 to 4 days, however, it was found that colonies of dividing cells arose in some of the cultures, at 2 to 4 weeks p.i. This occurred in all cultures infected with the mutant H I 8 and in a proportion of the cultures infected with the mutants F294 , H98 , N32 and FI27 (Table 2). The behaviour of these colonies was as described by Schw/3bel & Ahl (1972). They went through cyclical phases of c.p.e, and recovery, and eventually half of the infected cultures were lost. For two of the cultures infected with H98, however, and all 5 of the cultures infected with H 18, the colonies eventually grew out to form a confluent monolayer. This occurred at 6 to 8 weeks after the initial infection. These confluent cultures were split i/2 every 2 to Io days. They went through cyclical phases of c.p.e, and recovery, but remained viable as long as they were split at a ratio no less than I/2, immediately on reaching confluence. At the fifth passage after infection, the cells were frozen in liquid nitrogen and the medium frozen in samples at --7o °C. The experiments described below were all performed on cells at passages 5 to ~o p.i. Properties of ts mutants able to establish persistent infection Table 2 indicates that three R N A ÷ mutants and two R N A - mutants initiated colony formation, but that only two mutants (H98 and H 18) established stable persistent infections. Since one of these mutants was RNA-, the other R N A +, it was not immediately apparent what properties enabled these mutants to establish persistent infection. To clarify this situation, two further sets of experiments were performed.
20 4
G. J. A T K I N S T a b l e 3. Production o f mutant and revertant virus by monolayers infected at 39 ° C * Time after infection (h)
Yield assayed at 3o °C (p.f.u./ml/h)
Yield assayed at 39 °C (p.f.u./ml/h)
Revertant frequency
Fxo4
48-49 72-73
2.2 x IO5 I "8 x IO4
3"4 x IOs 8.2 x IOa
l "5 0. 5
F294
48-49 72-73
1.9 x Ios 4"4 x IO5
7"2 x 1oa 7'2 x I01
3"8 x lO-3 1"6 x 10-~
H98
48-49 72-73
8"6 x lOs 1"4 x 104
I "3 x lO4 5.0 X lo I
I "5 x 1o-z 3"5 x lo -3
N2
48-49 72-73 48-49 72-73
~'2 x 4"2 × 3"4 × 5"0 x
8"4x lo 5 5"4 x IO5 I '6 x lo 5 5"8 X IOs
0"7 I'3 0"5 1"2
A82
48-49 72-73
3"4 x IO# 3"6 X 1oB
2'8 x loS~ 7"4 x IO5~
0"8 0"2
AI2O
48-49 72-73 48-49 72-73
3"4 x 3"7 × 2-8 x 2' 9 x
3"4 x 4"7 x r .o × 3"2 ×
A93
48-49 72-73
3"2 × lo 4 9"6 x Io a
3"3 × Io4 1"4 × lo 4
HI8
48-49 72-73 48-49 72-73 48-49 72-73 48-49 72-73
4"6 × Io a 1"4 X IOt I '9 × IO4 7"4 × Ioa 3'2 X 10s 1"4 × IO4 i-6 x lo 5 5"0 x IO~
3"4 × Io 1 u:~ i-6 x Io t 9'2 x lOs 3"3 × 104 I'9 X IO~ 2.2 × io a 3"2 x ~OI
Mutant
N7
F36
N32 F127 Wild-type
Ioe IO~ IO5 IOs
IO~ Io4 IO~ 104
xos IO4 IO~ 10a
I'O i- 3 3"6 x Io ~ O-I I.o I-5 7"4 x 1o-a u~ 0"8 I "2 O'I 1"4 l "4 0"6
* Monolayers were infected at an input multiplicity of I p.f.u./celi. i" Pin-point plaques. :~ u = undetectable.
F i r s t l y , t h e yield o f v i r u s p r o d u c e d b y c u l t u r e s i n f e c t e d a t a n i n p u t m u l t i p l i c i t y o f I p . f . u . / c e l l w a s a s s a y e d a t 39 a n d 30 °C, a t 48 t o 49 h a n d 72 t o 73 h a f t e r i n f e c t i o n . A s s h o w n in T a b l e 3, t w o R N A - m u t a n t s ( F 2 9 4 a n d H 9 8 ) s h o w e d l o w r e v e r s i o n (as m e a s u r e d b y e . o . p , a t 39 ° C c o m p a r e d t o 3o °C) a t e a r l y s t a g e s ( u p t o 73 h ) p.i. T h e s a m e t w o m u t a n t s w e r e a b l e t o i n i t i a t e c o l o n y f o r m a t i o n in i n f e c t e d c u l t u r e s . O t h e r R N A - m u t a n t s s h o w e d h i g h reversion and inability to initiate colony formation. The e s t a b l i s h m e n t o f stable persistence in t w o o f t h e c u l t u r e s i n f e c t e d w i t h H 9 8 a n d n o n e o f t h o s e i n f e c t e d w i t h F 2 9 4 m a y h a v e b e e n coincidental. This correlation between low reversion and ability to initiate colony f o r m a t i o n did n o t apply to RNA + mutants. Only one (HI8) of the three RNA ÷ mutants (HI8, N32, FI27) able to initiate colony f o r m a t i o n s h o w e d low reversion. E x p e r i m e n t s were therefore perf o r m e d t o d e t e c t i n t e r f e r e n c e b e t w e e n ts m u t a n t s a n d t h e w i l d - t y p e ( T a b l e 4). M o n o l a y e r s o f cells in 60 m m p l a s t i c d i s h e s w e r e i n f e c t e d w i t h t h e w i l d - t y p e a t a n i n p u t m u l t i p l i c i t y o f I p . f . u . / c e l l a n d w i t h ts m u t a n t s a t a n i n p u t m u l t i p l i c i t y o f l o p . f . u . / c e l l t o give m i x e d inf e c t i o n s a t 39 °C. Cells i n f e c t e d w i t h t h e w i l d - t y p e v i r u s a l o n e w e r e a l s o set u p . T h e v i r u s y i e l d p r o d u c e d b y t h e s e c u l t u r e s a t 4 t o 6 h a n d 6 t o 24 h a f t e r i n f e c t i o n w a s t h e n c o l l e c t e d a n d t i t r a t e d a t 39 ° C t o give t h e yield o f w i l d - t y p e virus. A s s h o w n in T a b l e 4, t h e R N A m u t a n t s s h o w e d only slight interference with the g r o w t h o f the wild-type. R N A + m u t a n t s
Ts mutants o f Sindbis virus
205
Table 4. Interference by ts mutants with the multiplication of the wild-type virus at 39 °C Interference ratio A.
Mutant FIo4 F294
4 to 6 h l "3 i .2
6 to 24 2.6 3"4
H98 N2 N7 A82 AI20
I" I I'I O'9 !'6 2'1
2"2 2"0 3"2 4"2 6'5
F36
2-2
Io
A93 HI8
2.1 4'0
!I 19
N32 FI27
4'8 7"4
83 I57
Table 5. Total yield and temperature-sensitivity of virus produced by persistently infected
cells at passage 5 Cell line BHK/HI8
BHK/H98
Isolate I
Total yield (p.f.u./1o 6 cells)* 3"4 × to4
E.o.p. (39 °/3o °C) of virus produced o-8
2
3 .2 × l O s
~ '3
3
5"4 × Io5
o'9
4
8-6 × Io s
I'o
5
I "9 × 107
0.6
I 2
7-2 x IO5 2"8 Y 106
I 'I 0" 5
* As assayed at 3o °C. in general showed a greater degree of interference. This interference was greatest in the 6 to 24 h yield samples. The three mutants HI8, N32 and FI27, able to initiate colony formation in infected cultures, showed the greatest degree of interference. It is concluded that to set up even a transient persistent infection, a mutant has to show either low reversion, or interference. The mutant H I 8 , which is able to initiate persistent infections with the greatest efficiency, exhibits both these properties.
Properties of virus produced by persistently infected cultures Fluids from the seven stable persistently infected cultures frozen at the fifth passage were plaque assayed at 39 ° and 30 °C (Table 5). All seven cultures produced virus but the yields varied from 3 × Io4 to 2 × Io 7 p.f.u./IO 6 cells. Also, the plaque size of this virus was generally smaller than that produced by the wild-type virus and more variable in size. It is also clear from Table 5 that this virus was no longer temperature-sensitive. The ability of this released virus to set up fresh persistent infections was also tested. Cells in 25 cm ~ plastic bottles were infected with fluids from the passage 5 cells (five monolayers per sample). F o r every sample tested, colonies developed in all five cultures 2 to 3 weeks p.i., although the cultures did show extensive c.p.e, initially. Thus the released virus had an enhanced ability to initiate persistent infection.
Superinfection of persistently infected cells Uninfected and persistently infected B H K cells were superinfected with wild-type virus and virus-specified R N A synthesis measured (Table 6). Virus yields were not measured in this experiment as the a m o u n t o f virus released by persistently infected cells was often
206
G. J. A T K 1 N S
Table 6. Virus-specified R N A synthesis in persistently infected cells superinfected with the wild-type virus Cell line BHK BHK/H 18 BHK/H98
Sample Uninfected Uninfected Infected Uninfected Uninfected Infected Uninfected Uninfected Infected
Presence or absence of actinomycin D + -+ + -+ + -+
ct/min 180 5z 992 18060 348 26594 16t 8 258 I o 164
312
equivalent to that released by wild-type virus at early times p.i. Since the number of cells adhering to the dishes in these experiments was variable for persistently infected cells and large numbers of rounded cells were often present in the cultures, R N A synthesis was also measured in uninfected cells in the absence of actinomycin D. This incorporation gave a measure of the number of viable cells present. Even allowing for this, it is clear that virusspecified R N A synthesis is substantially reduced on superinfection of persistently infected cells compared to normal B H K cells. DISCUSSION
Previous studies (reviewed by Preble & Youngner, I975; Rima & Martin, I976; Holland & Levine, I978) have postulated that ts mutants, DI particles or interferon may be involved in the maintenance or establishment of persistent infection. This study indicates that the necessary condition for the establishment of persistent infection by ts mutants is an inhibition of the rate of virus multiplication in the early stages of infection. This may be achieved either by ts mutants having low reversion or by ts mutants interfering with the multiplication of wild-type revertants. It is possible that DI particles or interferon may act in the same way to establish persistent infection. In this context, this study may be compared with that of Schw~bel & Ahl 0972) who were able to establish persistent infection in the same cells using wild-type virus. These authors state, however, that the stocks of virus used in their experiments were at the fourteenth passage in chick embryo cells. It is possible that these virus stocks contained appreciable quantities of DI particles and that this was responsible for their ability to establish persistent infection. In the present study, virus stocks used to establish persistent infection were derived from cloned virus and were therefore unlikely to contain large quantities of DI particles (Bruton & Kennedy, I976). In cultures where reduced virus multiplication or interference has occurred, a few cells survive and divide t o form colonies. The resistance of the surviving cells to virus cytopathogenicity is variable, since the cultures exhibit cycles of c.p.e, and recovery. Thus the conclusion that can be drawn is that an inhibition of virus multiplication early in infection leads to the selection of a population of cells exhibiting a cyclical state of resistance to cytopathogenicity. The virus released from these cells is no longer temperature-sensitive, so it is clear that the ts mutation, although important in the establishment of persistent infection, is not required for its maintenance. The nature of the mechanism maintaining persistent infection in this system remains unclear. The early restriction of virus multiplication occurring in persistently infected cells resembles the interferon induced antiviral state. However, preliminary experiments have
Ts mutants of Sindbis virus
207
failed to detect interferon in any of the passage 5 fluids from persistently infected cells. There is one report that BHK-21 cells do not produce interferon (Taylor-Papadimitriou & Stoker, I97 I) but Wiktor & Clark (~ 972) in their study of BHK-21 cells persistently infected with rabies virus, were able to detect the transient production of small amounts of interferon. Thus it is possible that the persistently infected cells isolated in the present study may transiently produce small amounts of interferon, or that the initial reduced rate of virus multiplication during the establishment of persistent infection has resulted in the selection of a small subpopulation of cells capable of producing interferon and sensitive to its action. It is also possible that the maintenance of persistent infection in these cells results from the action of DI particles. Preliminary experiments have indicated that they do indeed produce DI particles. Although co-infection of sensitive cells with DI particles and standard virus reduces infectious virus production, total virus-specified RNA synthesis is not inhibited (Weiss & Schlesinger, 1973). However, it is possible that pre-existing DI RNA in persistently infected cells may inhibit RNA synthesis by the standard virus. Studies on the mechanism of maintenance of persistent infection in these cells are continuing. 1 thank the Medical Research Council of Ireland and the Irish Cancer Society for financial support, and Mrs Lynda Winston for excellent technical assistance. REFERENCES ATKINS, G. J. 0976). T h e effect o f infection with Sindbis virus a n d its temperature-sensitive m u t a n t s o n cellular protein a n d D N A synthesis. Virology 7 r, 593-597. A'rKINS, G. S. (1977). Cytopathogenicity of temperature-sensitive m u t a n t s of Sindbis virus. FEMS Microbiology Letters 2, 51-55. ATKINS, G. J., SAMtJELS, J. & KENNEDY, S. I. 1". (1974). Isolation a n d preliminary characterisation o f temperaturesensitive m u t a n t s o f Sindbis virus strain AR339. Journal of General Virology 25, 371-38o. 8RtJTON, C. J. & KENNEDY, S. I. T. (I976). Defective interfering particles of Semliki Forest virus; structural differences between s t a n d a r d virus a n d defective-interfering particles. Journal of General Virology 3 x, 383-396. BRZESKI, H. & KENNEDY, S. I. T. (I978). Synthesis o f alphavirus-specified R N A . Journal of Virology 25, 630-64o. BRZESKI, n., CLEGG, J. C. S., ATKINS, G. J. & KENNEDY, S. 1. "r. (I978). Regulation of the synthesis o f Sindbis virus-specified R N A : role o f the virion core protein. Journal of General Virology 38, 461-47o. EATON, 8. T. 0977). Evidence for the synthesis o f defective interfering particles by Aedes albopictus cells persistently infected with Sindbis virus. Virology 77, 843-848. HOLLAND, J. J. & LEVINE, A. 0978). M e c h a n i s m s of viral persistence. Cell x4, 447-452. INGLOX, A. O., ALBIN, M. & CHtJDZIO, T. 0973). Persistent infection of m o u s e cells with Sindbis virus: role o f virulence o f strains, auto-interfering particles a n d interferon. Journal of General Virology 2o, [o5-I IO. MAcPSERSON, L A. & STOgER, M. G. P. 0962). P o l y o m a t r a n s f o r m a t i o n o f h a m s t e r cell clones - - an investigation o f genetic factors affecting cell competence. Virology i6, 147-154. PREBLE, O. T. & ¥OtJNGNER, J. S. 0975): Temperature-sensitive viruses a n d the etiology o f chronic a n d ina p p a r e n t infections. Journal of Infectious Diseases I3 x, 467-473RIMA, 8. & r,IARTIr~, S. J. (1976). Persistent infection o f tissue culture cells by R N A viruses. Medical Microbiology & Immunology x62, 89-118. SCHW6BEL, S. ,~ AHL, R. (I972). Persistence o f Sindbis virus in BHK-21 cell cultures. Archives of Virology 38, I-IO. S~tENK, "r. E., KOSttELNVK, K. A. & STOLLAR, V. 0974)- Temperature-sensitive virus f r o m Aedes albopictus cells chronically infected with Sindbis virus. Journal of Virology x3, 439-447. TAYLOR-PAPADIMITRIOU, J. & STOKER, M. (1971). Effect o f interferon o n s o m e aspects of t r a n s f o r m a t i o n by p o l y o m a virus. Nature New Biology ~'3o, I I4-117. WEISS, a. & SCHLESINGER, S. 0973). Defective interfering passages o f Sindbis virus: chemical composition, biological activity a n d m o d e o f interference. Journal of Virology x~,, 86z-87~ WlI,:TOR, "r. j. & CLARiC, H. F. 097Z)- C h r o n i c rabies virus infection of cell cultures. Injection and lmmunity 6, 988--995.
(Received 15 February I979)
