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Further studies on the infection of pig-kidney cells by foot-and-mouth disease virus
768
BIOCHIMICA ET BIOPHYSICA ACTA
F U R T H E R STUDIES ON T H E INFECTION OF P I G - K I D N E Y CELLS BY FOOT-AND-MOUTH DISEASE VIRUS F. B R O W N ,
B. C A R T W R I C - H T AND D O R E E N
L. S T E W A R T
Research Institute, Animal Virus Diseases, Pirbright, Surrey (Great Britain) (Received A u g u s t 2 i s t , 1961)
SUMMARY Foot-and-mouth disease virus attaches to pig-kidney tissue-culture cells in the presence of calcium ions. At low temperature (2-4 ° ) the virus does not penetrate the cells but remains on the surface. The viral ribonucleic acid is not released from the virus and most of the attached virus can be eluted from the cells with EDTA. At 37 ° the virus penetrates the cell and is broken down into its nucleic acid and protein components. Disruption of the virus can also be achieved b y cells which have been broken down by freezing and thawing. Virus also attaches to cells which have been killed with merthiolate or iodoacetate but penetration does not occur.
INTRODUCTION
Current ideas on the mechanism by which cells are infected by animal viruses suggest that viral nucleic acid is released from the infecting virus at some stage in the process. At present information on the precise mechanism appears to be lacking but it seems that two main alternatives are possible: (a) the virus is engulfed and breaks down within the cell or (b) the virus disintegrates at the cell surface, only the nucleic acid entering the cell, as in the infection of bacteria by phage 1. It is unlikely that different viruses infect their respective hosts by identical mechanisms, especially as the process is under the control of a viral enzyme in some instances (e.g. influenza) but not under such control with most other viruses. Even for one virus, influenza, evidence has been presented for each of the two alternatives indicated above. Thus FAZEKAS DE ST. GROTH2 favours the first alternative, calling the engulfment process "viropexis", whereas HOYLE et al.S, 4 have concluded that the virus breaks down at the cell wall, only the nucleic acid entering the cell. The recent work by McLAREN et al. 5, eand JOKLIK AND DARNELL~ on the adsorption of poliovirus by Hela cells does not allow any conclusion to be drawn about the site of breakdown of the virus. In a previous paper s the events which occurred following the addition of footand-mouth disease virus to pig-kidney tissue-culture cells were described. It was shown that, although more than 9 ° % of the virus infectivity was lost, all the ii,fective RNA contained in the virus inoculum could be recovered from the cells. The experiments described here, which are an extension of this work, were carried out in an attempt to obtain a fuller understanding of the mechanism of infection. Biochim. Biophys. Acta, 55 (I962) 768-774
INFECTION BY" FOOT-AND-MOUTH DISEASE VIRUS
769
MATERIALS AND METHODS
Titration methods The methods used for the titration of virus and for the extraction and estimation of infective RNA were described fully in a previous paper s. In all virus titrations the sample to be examined was first diluted to lO -1 in o.I % sodium dodecyl sulphate in order to disintegrate cells and the subsequent dilutions made in o.04 M phosphate buffer or phosphate-buffered saline, p H 7.4. This concentration of sodium dodecyl sulphate has no effect on the infectivity of foot-and-mouth disease virus suspensions. The titrations were made in unweaned mice 9 or occasionally b y plaque titration on pig-kidney monolayers. All estimations of infective RNA were done b y observing the cytopathic effects on pig-kidney monolayers. For the titration of complement fixing antigen the method used was that described b y BROOKSBY 1°, in which dilutions of complement are reacted with constant amounts of antigen and antiserum.
Virus and tissue-culture cells Virus of strain 997 (Waldmann type C), obtained b y serial passage in pig-kidney cell monolayers, was used in all experiments. Tissue-culture cells, provided b y the tissue-culture unit of this Institute, were used as described earlier 8. In general lO7 ID5o of virus were added to IO7 cells or the debris prepared from this number of cells.
Estimation o/ in/ective centres Suspensions of cells, obtained from monolayers grown in Roux flasks b y treatment with a E D T A - t r y p s i n mixture (o.oi % of each) were washed in phosphatebuffered saline, p H 7.4, centrifuged at 2000 rev./min and the deposited cells suspended in virus at the desired temperature. After 15 rain at 37 ° or more prolonged periods at 2 ° the virus inoculum was removed b y centrifuging at 2000 rev./min and the cells resuspended in o.15 M acetate buffer, p H 6.0 for 30 sec. This procedure removed most of the extra-cellular virus without causing damage to the cells. The acetate buffer was removed b y centrifugation and the ceils resuspended in phosphate-buffered saline, p H 7.4. The cells in the suspension were then counted and suitable dilutions inoculated either (a) intraperitoneally into 7-day-old unweaned mice or (b) on to pig-kidney cell monolayers, overlaid with agar and the number of plaques formed at 48 h counted after staining with neutral red or (c) into cups containing IOs uninfected pig-kidney tissue-culture cells suspended in Hanks' balanced salt solution containing 2 % calf serum, 0.5 % lactalbumiu hydrolysate, o.oi % yeast extract and 0.002 % phenol red, overlaid with liquid paraffin and incubated for 72 h at 37 °. In this last method (the metabolic inhibition test11), the presence of an infected cell in the inocuhim led to the production of virus which infected the other cells and so prevented the formation of acid which occurs with uninfected cells. RESULTS
Estimation o~ number o/ cells in/ected by virus inocula Previous workers with foot-and-mouth disease virus have measured the number of cells which are infected (i.e. become virus producers) when mixed with the virus either b y inoculating dilutions of the washed cells into unweaned mice TM or on to Biochim. Biophys. Acta, 55 (I962) 768-774
770
F.
BROWN,
B. C A R T W R I G H T ,
D.
L.
STEWART
pig-kidney cell monolayers 13 and subsequently counting the number of mice which died or the number of plaques which were formed. Both methods indicated that only a small proportion of the cells were infected when the virus-cell ratio was unity and even when this ratio was greater than IO it appeared that not all the cells became virus producers. We have confirmed these results but have found that the use of the metabolic inhibition test n gives higher values. For example, when the virus-cell ratio was I, the mouse and plaque tests indicated that only about IO % of the cells become virus producers, whereas the metabolic inhibition test showed that almost one half of the cells were infected (Table I). In case the failure of the cells to metabolize and TABLE
I
ESTIMATION OF NUMBER OF PIG-KIDNEY TISSUE-CULTURE CELLS INFECTED BY FOOT-AND-MOUTH DISEASE VIRUS AT 3 7 °
Per cent cells injected as estimated by Virus/cell ratio
Mouse titration
I
IO
Plaque method
9
Metabolic inhibition t e s t
6
48
II
--
I00
24
--
IOO
therefore not produce acid (so appearing positive) was due to some metabolic inhibitor other than the virus which the infected cells produced, the cups containing the cells were tested for the presence of virus by inoculation into 7-day-old unweaned mice. All the cups which remained above pH 7.2, and consequently were counted positive, were shown to contain infective virus.
Effect o/ temperature on the in/ection o/ pig-kidney cells It was stated in an earlier paper s that the addition of foot-and-mouth disease virus to the cells at 2° led, as at 37 °, to removal of most of the virus from the supernatant fluid and to a considerable overall loss of virus infectivity. Although as much infective R N A could be obtained from these cells as from the virus inoculum, the virus titre of the cells was only I-IO % of that of the inoculum, In addition, if the cells were first ground in a mortar at 2 ° before extracting with phenol, the yield of infective RNA was considerably lower than that obtained from the intact cells (Table II). It thus TABLE
II
DISTRIBUTION OF VIRUS INFECTIVITY AND INFECTIVE R N A KIDNEY
AFTER ADDITION OF VIRUS TO PIG
CELLS
Virus (log I D s o / m l ) 2°
Initial virus Supernatant Cells Cells g r o u n d wit h r i b o n u c l e a s e Ceils washed at p H 6.0
In/eetive R N A (log I D s o / m l )
37 °
2°
37 °
6.5
6.5
1.5
1. 5
5 .o
4.8
None detected
None detected
5 .o
4.5
1.7
1.5
--
None detected
None detected
0. 3
None detected
. i.~
o. 5
Biochim.
Biophys.
Acta,
55
(1962)
768-774
771
INFECTION BY FOOT-AND-MOUTH DISEASE VIRUS
appeared that virus was being broken down at 2 ° in the same way as at 37 ° . However, the number of cells infected under these conditions was found to be less than 1 % of those which became infected b y a similar virus inoculum at 37 °. Furthermore, cells mixed with virus at 2 ° and then washed with o.15 M acetate buffer, p H 6.0, failed to yield any infective RNA on extraction with phenol. This result is in direct contrast to that obtained with cells infected at 37 ° as at this temperature the yield of infective RNA from acetate-washed cells is similar to that obtained from infected cells washed with phosphate-buffered saline (Table II). These results provided evidence that the virus which had attached to the cells at 2 ° had not penetrated the cell wall and was still inactivated b y acid treatment.
Recovery o/ adsorbed virus ]rom calls in/ected at 2 ° As the cells apparently did not become infected at 2 °, although the virus was adsorbed from the supernatant fluid, it seemed possible that the attachment at 2 ° was reversible, as in the case of Coxsackie virus to human amnion cells 14. Attachment of the virus of foot-and-mouth disease to pig-kidney cells does not occur if the virus and cells are suspended in calcium and magnesium-free phosphate-buffered saline. Addition of 10 -3 M calcium ions is sufficient to allow attachment (Table III). It
INFLUENCE
OF
CALCIUM
IONS
TABLE
III
ON
ATTACHMENT
THE
OF
VIRUS
TO
CELLS
"[.rf~ru s
(log ID5o/ral)
Initial virus Supernatant Initial virus Supernatant Initial virus Supernatant
in Ca a n d Mg-free saline f r o m cells in Ca a n d Mg-free s a l i n e + x o - * M Ca f r o m cells in Ca a n d Mg-free s a l i n e + I o -8 M Ca f r o m cells
5.4 5.3 5.4 5.6 5.4 4.4
was considered that the addition of a chelating agent such as E D T A would release virus which had already attached to pig-kidney cells if the virus was still on the outside of the cells. This elution of virus was in fact effected as considerable recovery of the attached virus, measured by both infectivity determinations and complementfixation tests, could be achieved b y shaking the intact cells at 2 ° with I o - 3 M E D T A (Table IV). This concentration of E D T A does not have any effect on pig-kidney TABLE RECOVERY
OF
VIRUS
ATTACHED
TO
IV
PIG-KIDNEY
AT
2 ° AND
37 °
Complemeng-tixing activity (ml z /3o complement ]ixed by z ml antigen)
Virus
(log IDso/ml)
Initial v i r u s Supernatant Cells C e l l s + i o -~ M E D T A
CELLS
~o
37 °
ao
7.0 5.5 5.o 7 .o
7.0 5.3 4.8 4.9
0.862 o.214 o.35o* o.519
37 °
0.862 o.171 None detected None detected
" T e s t e d a f t e r a d d i n g M e r t h i o l a t e (see t e x t ) . Biochim. Biophys. Aaa,
55 (1962) 7 6 8 - 7 7 4
772
F. BROWN, B. CARTWRIGHT, D. L. STEWART
tissue-culture cells because cells infected at 37 ° and subsequently washed with E D T A produce as much virus as control cells washed with phosphate-buffered saline. When cells are infected at 37 °, subsequent washing with EDTA does not lead to the recovery of any virus, indicating that in this case the viral antigen is no longer attached to the cell surface. I t was shown in earlier experiments 8 that ether extraction of cells infected at 37 ° led to the recovery of the 7-m# non-infective antigen of the virus but infective virus was not recovered. Similar treatment of cells to which virus had been added at 2 ° led to the recovery of a considerable proportion of the adsorbed infective virus, confirming that disruption of the virus particle had not occurred. In this case, however, the ether disrupts the cells so the result does not provide information regarding the site to which the virus is attached. The complement-fixing activity of cells which have been mixed with virus at 2 ° cannot be measured directly as the test is normally carried out at 37 °. To overcome this difficulty, cells which had been mixed with virus at 2 ° were suspended at 2 ° in lO -8 M Merthiolate (sodium ethylmercurithiosalicylate) for a few hours before the complement-fixation test. This procedure kills cells and so prevents engulfment of virus (see below). Cells treated in this way had a considerable amount of complementfixing antigen on their surface, in contrast to cells infected at 37 ° (Table IV).
E//ect o/ cell debris on virus Debris was prepared from pig-kidney cells b y freezing and thawing suspensions at --7 °0 and + 2 0 ° respectively. Staining these preparations with methylene blue showed that no intact cells remained although a considerable number of nuclei was observed. When the disintegrated cells were added to virus suspensions at 2 ° or at 37 °, considerable loss of infectivity was encountered similar to the losses with intact cells. Extraction of the virus-debris mixtures with phenol resulted in variable recoveries of infective RNA. In most experiments the amount of infective RNA which was recovered was proportional to the residual virus titre, indicating that breakdown of the virus had occurred and the released RNA had been hydrolysed by the ribonuclease in the medium (Table V). In some experiments, however, loss of virus titre was not accompanied by any decrease in the amount of infective RNA which could be recovered. On these occasions the "lost" virus could be recovered by addition of E D T A to the virusdebris mixture. Similar recovery of virus could not be achieved from those mixtures which gave low recoveries of RNA on extraction with phenol. TABLE V INACTIVATION
OF VIRUS
AND
INFECTIVE
RNA
BY
CELL DEBRIS
(log I D so /rnl)
In/eaive R N A (log I D so lrnt)
0.5 5.0
1.5 o.2
5.0 5.2
1.5
Virus
Initial v i r u s Virus + freeze-thawed debris Virus + freeze-thawed debris + 10 -2 M E D T A Virus + g r o u n d debris Virus + g r o u n d debris + IO -s M E D T A
-
6. 3 Biochim.
-
Biophys.
Acta,
-
-
55 (1962) 768-774
773
I N F E C T I O N BY F O O T - A N D - M O U T H D I S E A S E V I R U S
Debris prepared from cells b y grinding in a mortar also inactivated virus but the virus could be recovered b y adding E D T A and the yield of RNA was the same as that obtained from the virus only. Similarly, debris obtained b y grinding freezethawed debris attached virus reversibly but did not disintegrate the virus particle. Presumably grinding cells releases a substance which can disintegrate virus already present on the cells. This substance must be very unstable as the addition of ground debris to virus does not lead to disintegration of virus although attachment occurs. This instability m a y account for the variable results obtained with different preparations of freeze-thawed debris. An interesting observation which m a y be of importance in this connection was made in the phenol extraction of cells and the debris preparations. I n t a c t cells and freeze-thawed debris invariably gave a thin layer of protein at the water-phenol interface whereas ground debris did not, all the protein being at the b o t t o m of the phenol layer.
Location o/ substance in cells which disintegrates virus When virus was added to cells at 2 ° the virus attached but could be removed b y E D T A or ether. Extraction of the cells with phenol yielded as much infective RNA as could be obtained from the virus. If the infected cells were first ground in a mortar at 2 ° in phosphate-buffered saline containing 0.I/~g ribonuclease/ml, taking care t h a t the temperature did not rise, subsequent extraction of the ground cells gave a poor recovery of infective RNA (Table II). The addition of E D T A to these ground cells did not increase the virus titre, in contrast to the increase obtained when E D T A was added to the intact cells. This result suggests that the mechanism for disintegrating virus into its RNA and protein components is situated inside the cell and is released when the cells are ground.
E//ect o/ adding virus to dead cells Cells were killed b y treatment for I h at room temperature with 10 -3 M Merthiolate or 10 -3 M iodoacetate. After this treatment the cells did not metabolize to produce acid in a medium containing glucose nor did they produce any virus after infection. They adsorbed virus, however, to the same extent as untreated cells. The figures in Table VI show typical results obtained with cells killed with Merthiolate. Similar results were obtained with cells killed with iodoacetate. In contrast to normal TABLE DISTRIBUTION
OF INFECTIVE
VI
VIRUS, VIRAL PROTEIN AND KIDNEY CELLS KILLED WITH
Virus
(log ID5o/ml ) Initial virus
6.8
Supernatant
5.0
Cells Cells g r o u n d
with ribonuclease
Cells w a s h e d a t p H 6 . o C e l l s " { - 10 -2 M E D T A
4.5 -o. 5 6.8
R N A AFTER ADDITION MERTHIOLATE Infective R N A (log IDso/ml) I. 7 None detected i .7 1.8 None detected --
Biochim. Biophys. Acta,
OF VIRUS TO PIG-
Complement-fixing activity (ml r [3o complement fixed by z ml antigen) 0.760 o.i8o o.45t) ----
55 ( z 9 6 2 ) 7 6 8 - 7 7 4
774
F. BROWN, B. CARTWRIGHT, D. L. STEWART
cells, however, the a d s o r b e d virus was n o t b r o k e n down to R N A a n d viral p r o t e i n as it could be r e c o v e r e d c o m p l e t e l y b y a d d i t i o n of E D T A to the cells. Also, killed cells to w h i c h virus h a d been a d d e d , a l t h o u g h yielding as m u c h R N A as the i n o c u l a t e d virus, d i d n o t yield a n y R N A if t h e y were first washed with o.15 M acetate, p H 6.0. This i n d i c a t e d t h a t t h e virus was still on t h e outside of t h e cell a n d c o m p l e m e n t - f i x a tion tests confirmed this supposition. A d d i t i o n a l l y , t h e c e l l - v i r u s c o m p l e x y i e l d e d as m u c h infective R N A even a f t e r grinding w i t h ribonuclease, confirming t h a t t h e virus h a d n o t b r o k e n down. DISCUSSION There is l i t t l e i n f o r m a t i o n to show h o w viruses infect a n i m a l cells. A d s o r p t i o n of t h e virus to t h e cell w o u l d a p p e a r to be a necessary first s t e p a n d c u r r e n t ideas on the l a t e r stages of t h e infection process f a v o u r t h e view t h a t t h e infective nucleic acid is released from t h e virus. T h e site at which this t a k e s place, in the case of p i g - k i d n e y tissue-culture cells a n d f o o t - a n d - m o u t h disease virus, a p p e a r s to be within t h e cell. Thus a d s o r p t i o n of t h e virus to cells can occur as efficiently at 2 ° as a t 37 ° b u t t h e virus does n o t e n t e r t h e cell at the lower t e m p e r a t u r e as it can be released b y E D T A w i t h o u t d i s r u p t i n g t h e cell. The a d d i t i o n of s o d i u m d o d e c y l s u l p h a t e to t h e c e l l virus c o m p l e x a t 2 ° does n o t release the virus a l t h o u g h the cells are c o m p l e t e l y d i s r u p t ed. I t w o u l d a p p e a r t h a t t h e r e c e p t o r site on the cell remains a t t a c h e d to t h e virus a n d so p r e v e n t s it infecting o t h e r cells. A t 2 ° no d e t e c t a b l e infective R N A enters t h e cell nor do a n y cells become infected. If t h e v i r u s - c e l l complex is g r o u n d up, however, k e e p i n g t h e t e m p e r a t u r e low, t h e n virus can no longer be recovered b y E D T A ext r a c t i o n , n o r can infective R N A be o b t a i n e d from t h e debris. I t thus a p p e a r s t h a t the m e c h a n i s m for releasing the R N A from t h e virus is n o t on t h e cell surface b u t inside the ceJ1. I t is p r o b a b l y significant t h a t in a few e x p e r i m e n t s in which t h e v i r u s - c e l l c o m p l e x is h e l d at 2 ° for p r o l o n g e d periods, r e c o v e r y of infective R N A is low, indicating t h a t t h e s u b s t a n c e s in t h e cell which are responsible for virus d i s r u p t i o n m a y h a v e leached out of the cell a n d d i s r u p t e d t h e virus on t h e cell surface. Debris prep a r e d from cells b y freeze-thawing can d i s r u p t virus into R N A a n d p r o t e i n even at low t e m p e r a t u r e . I t is likely, therefore, t h a t a l t h o u g h a t t a c h m e n t is efficient at 2 ° , t r a n s p o r t of t h e virus into t h e cell does n o t occur. T h e m e t h o d b y which f o o t - a n d - m o u t h disease virus infects p i g - k i d n e y cells a p p e a r s to be b y an engulfment process which does n o t o p e r a t e at low t e m p e r a t u r e s . This view w o u l d seem to be confirmed b y t h e o b s e r v a t i o n t h a t killed cells, which would n o t be e x p e c t e d to engulf the virus, can a d s o r b t h e virus w i t h o u t releasing its R N A . REFERENCES 1 A. D, HERSHEY AND M. CHASE, J. Gen. Physiol., 36 (1952) 39S. FAZEKASDE ST. GROTH, Nature, 162 (1948) 294. 3 L. HOYLE AND W. FRISCH-NIGGEMEYER, J. Hyg., 53 (1955) 474. 4 L. BOYLE AND N. B. FINTER,
J. Hyg.,
55 (1957) 290.
5 L. C. McLAREN, J. J. HOLLANDAND J. "i'. SYVERTON,J. Exptl, Med., lO9 (1959) 475-
MCLAREN, J. Exptl. Med., lO9 (1959) 487 • W. K. JOKLIK AND J. E. DARNELL, Virology, 13 (1961) 4398 F. BROWN, B. CARTWRIGHTAND D. L. STEWART,Biochim. Biophys. Acta, 47 (1961) 172. 0 H. H. SKINNER, Proc. Roy. Soc. Med., 44 (1951) l°4110 j. B. BROOKSBY, Agr. Research Council Rept., Ser. No. I2 (1952). 11 W. B. MARTINAND W. C*. CHAPMAN, Research Vet. Sci., 2 (1961) 53. 12 S. F. CARTWRIGHT, T. W. F. PAY AND W. M. HENDERSON, J . Gen. Microbiol., 16 (1957) 73 TM 13 R. F. SELLERS, Arch. Virus/orsch., 9 (1959) 621. 14 L. C. MCLAREN, J. J. HOLLANDAND J. T. SYVERTON, J. Exptl. Med., II2 (196o) 581. 6 j . j . HOLLAND AND L. C.
Biochim. Biophys. Aaa, 55 (1962) 768-774
BIOCHIMICA ET BIOPHYSICA ACTA
F U R T H E R STUDIES ON T H E INFECTION OF P I G - K I D N E Y CELLS BY FOOT-AND-MOUTH DISEASE VIRUS F. B R O W N ,
B. C A R T W R I C - H T AND D O R E E N
L. S T E W A R T
Research Institute, Animal Virus Diseases, Pirbright, Surrey (Great Britain) (Received A u g u s t 2 i s t , 1961)
SUMMARY Foot-and-mouth disease virus attaches to pig-kidney tissue-culture cells in the presence of calcium ions. At low temperature (2-4 ° ) the virus does not penetrate the cells but remains on the surface. The viral ribonucleic acid is not released from the virus and most of the attached virus can be eluted from the cells with EDTA. At 37 ° the virus penetrates the cell and is broken down into its nucleic acid and protein components. Disruption of the virus can also be achieved b y cells which have been broken down by freezing and thawing. Virus also attaches to cells which have been killed with merthiolate or iodoacetate but penetration does not occur.
INTRODUCTION
Current ideas on the mechanism by which cells are infected by animal viruses suggest that viral nucleic acid is released from the infecting virus at some stage in the process. At present information on the precise mechanism appears to be lacking but it seems that two main alternatives are possible: (a) the virus is engulfed and breaks down within the cell or (b) the virus disintegrates at the cell surface, only the nucleic acid entering the cell, as in the infection of bacteria by phage 1. It is unlikely that different viruses infect their respective hosts by identical mechanisms, especially as the process is under the control of a viral enzyme in some instances (e.g. influenza) but not under such control with most other viruses. Even for one virus, influenza, evidence has been presented for each of the two alternatives indicated above. Thus FAZEKAS DE ST. GROTH2 favours the first alternative, calling the engulfment process "viropexis", whereas HOYLE et al.S, 4 have concluded that the virus breaks down at the cell wall, only the nucleic acid entering the cell. The recent work by McLAREN et al. 5, eand JOKLIK AND DARNELL~ on the adsorption of poliovirus by Hela cells does not allow any conclusion to be drawn about the site of breakdown of the virus. In a previous paper s the events which occurred following the addition of footand-mouth disease virus to pig-kidney tissue-culture cells were described. It was shown that, although more than 9 ° % of the virus infectivity was lost, all the ii,fective RNA contained in the virus inoculum could be recovered from the cells. The experiments described here, which are an extension of this work, were carried out in an attempt to obtain a fuller understanding of the mechanism of infection. Biochim. Biophys. Acta, 55 (I962) 768-774
INFECTION BY" FOOT-AND-MOUTH DISEASE VIRUS
769
MATERIALS AND METHODS
Titration methods The methods used for the titration of virus and for the extraction and estimation of infective RNA were described fully in a previous paper s. In all virus titrations the sample to be examined was first diluted to lO -1 in o.I % sodium dodecyl sulphate in order to disintegrate cells and the subsequent dilutions made in o.04 M phosphate buffer or phosphate-buffered saline, p H 7.4. This concentration of sodium dodecyl sulphate has no effect on the infectivity of foot-and-mouth disease virus suspensions. The titrations were made in unweaned mice 9 or occasionally b y plaque titration on pig-kidney monolayers. All estimations of infective RNA were done b y observing the cytopathic effects on pig-kidney monolayers. For the titration of complement fixing antigen the method used was that described b y BROOKSBY 1°, in which dilutions of complement are reacted with constant amounts of antigen and antiserum.
Virus and tissue-culture cells Virus of strain 997 (Waldmann type C), obtained b y serial passage in pig-kidney cell monolayers, was used in all experiments. Tissue-culture cells, provided b y the tissue-culture unit of this Institute, were used as described earlier 8. In general lO7 ID5o of virus were added to IO7 cells or the debris prepared from this number of cells.
Estimation o/ in/ective centres Suspensions of cells, obtained from monolayers grown in Roux flasks b y treatment with a E D T A - t r y p s i n mixture (o.oi % of each) were washed in phosphatebuffered saline, p H 7.4, centrifuged at 2000 rev./min and the deposited cells suspended in virus at the desired temperature. After 15 rain at 37 ° or more prolonged periods at 2 ° the virus inoculum was removed b y centrifuging at 2000 rev./min and the cells resuspended in o.15 M acetate buffer, p H 6.0 for 30 sec. This procedure removed most of the extra-cellular virus without causing damage to the cells. The acetate buffer was removed b y centrifugation and the ceils resuspended in phosphate-buffered saline, p H 7.4. The cells in the suspension were then counted and suitable dilutions inoculated either (a) intraperitoneally into 7-day-old unweaned mice or (b) on to pig-kidney cell monolayers, overlaid with agar and the number of plaques formed at 48 h counted after staining with neutral red or (c) into cups containing IOs uninfected pig-kidney tissue-culture cells suspended in Hanks' balanced salt solution containing 2 % calf serum, 0.5 % lactalbumiu hydrolysate, o.oi % yeast extract and 0.002 % phenol red, overlaid with liquid paraffin and incubated for 72 h at 37 °. In this last method (the metabolic inhibition test11), the presence of an infected cell in the inocuhim led to the production of virus which infected the other cells and so prevented the formation of acid which occurs with uninfected cells. RESULTS
Estimation o~ number o/ cells in/ected by virus inocula Previous workers with foot-and-mouth disease virus have measured the number of cells which are infected (i.e. become virus producers) when mixed with the virus either b y inoculating dilutions of the washed cells into unweaned mice TM or on to Biochim. Biophys. Acta, 55 (I962) 768-774
770
F.
BROWN,
B. C A R T W R I G H T ,
D.
L.
STEWART
pig-kidney cell monolayers 13 and subsequently counting the number of mice which died or the number of plaques which were formed. Both methods indicated that only a small proportion of the cells were infected when the virus-cell ratio was unity and even when this ratio was greater than IO it appeared that not all the cells became virus producers. We have confirmed these results but have found that the use of the metabolic inhibition test n gives higher values. For example, when the virus-cell ratio was I, the mouse and plaque tests indicated that only about IO % of the cells become virus producers, whereas the metabolic inhibition test showed that almost one half of the cells were infected (Table I). In case the failure of the cells to metabolize and TABLE
I
ESTIMATION OF NUMBER OF PIG-KIDNEY TISSUE-CULTURE CELLS INFECTED BY FOOT-AND-MOUTH DISEASE VIRUS AT 3 7 °
Per cent cells injected as estimated by Virus/cell ratio
Mouse titration
I
IO
Plaque method
9
Metabolic inhibition t e s t
6
48
II
--
I00
24
--
IOO
therefore not produce acid (so appearing positive) was due to some metabolic inhibitor other than the virus which the infected cells produced, the cups containing the cells were tested for the presence of virus by inoculation into 7-day-old unweaned mice. All the cups which remained above pH 7.2, and consequently were counted positive, were shown to contain infective virus.
Effect o/ temperature on the in/ection o/ pig-kidney cells It was stated in an earlier paper s that the addition of foot-and-mouth disease virus to the cells at 2° led, as at 37 °, to removal of most of the virus from the supernatant fluid and to a considerable overall loss of virus infectivity. Although as much infective R N A could be obtained from these cells as from the virus inoculum, the virus titre of the cells was only I-IO % of that of the inoculum, In addition, if the cells were first ground in a mortar at 2 ° before extracting with phenol, the yield of infective RNA was considerably lower than that obtained from the intact cells (Table II). It thus TABLE
II
DISTRIBUTION OF VIRUS INFECTIVITY AND INFECTIVE R N A KIDNEY
AFTER ADDITION OF VIRUS TO PIG
CELLS
Virus (log I D s o / m l ) 2°
Initial virus Supernatant Cells Cells g r o u n d wit h r i b o n u c l e a s e Ceils washed at p H 6.0
In/eetive R N A (log I D s o / m l )
37 °
2°
37 °
6.5
6.5
1.5
1. 5
5 .o
4.8
None detected
None detected
5 .o
4.5
1.7
1.5
--
None detected
None detected
0. 3
None detected
. i.~
o. 5
Biochim.
Biophys.
Acta,
55
(1962)
768-774
771
INFECTION BY FOOT-AND-MOUTH DISEASE VIRUS
appeared that virus was being broken down at 2 ° in the same way as at 37 ° . However, the number of cells infected under these conditions was found to be less than 1 % of those which became infected b y a similar virus inoculum at 37 °. Furthermore, cells mixed with virus at 2 ° and then washed with o.15 M acetate buffer, p H 6.0, failed to yield any infective RNA on extraction with phenol. This result is in direct contrast to that obtained with cells infected at 37 ° as at this temperature the yield of infective RNA from acetate-washed cells is similar to that obtained from infected cells washed with phosphate-buffered saline (Table II). These results provided evidence that the virus which had attached to the cells at 2 ° had not penetrated the cell wall and was still inactivated b y acid treatment.
Recovery o/ adsorbed virus ]rom calls in/ected at 2 ° As the cells apparently did not become infected at 2 °, although the virus was adsorbed from the supernatant fluid, it seemed possible that the attachment at 2 ° was reversible, as in the case of Coxsackie virus to human amnion cells 14. Attachment of the virus of foot-and-mouth disease to pig-kidney cells does not occur if the virus and cells are suspended in calcium and magnesium-free phosphate-buffered saline. Addition of 10 -3 M calcium ions is sufficient to allow attachment (Table III). It
INFLUENCE
OF
CALCIUM
IONS
TABLE
III
ON
ATTACHMENT
THE
OF
VIRUS
TO
CELLS
"[.rf~ru s
(log ID5o/ral)
Initial virus Supernatant Initial virus Supernatant Initial virus Supernatant
in Ca a n d Mg-free saline f r o m cells in Ca a n d Mg-free s a l i n e + x o - * M Ca f r o m cells in Ca a n d Mg-free s a l i n e + I o -8 M Ca f r o m cells
5.4 5.3 5.4 5.6 5.4 4.4
was considered that the addition of a chelating agent such as E D T A would release virus which had already attached to pig-kidney cells if the virus was still on the outside of the cells. This elution of virus was in fact effected as considerable recovery of the attached virus, measured by both infectivity determinations and complementfixation tests, could be achieved b y shaking the intact cells at 2 ° with I o - 3 M E D T A (Table IV). This concentration of E D T A does not have any effect on pig-kidney TABLE RECOVERY
OF
VIRUS
ATTACHED
TO
IV
PIG-KIDNEY
AT
2 ° AND
37 °
Complemeng-tixing activity (ml z /3o complement ]ixed by z ml antigen)
Virus
(log IDso/ml)
Initial v i r u s Supernatant Cells C e l l s + i o -~ M E D T A
CELLS
~o
37 °
ao
7.0 5.5 5.o 7 .o
7.0 5.3 4.8 4.9
0.862 o.214 o.35o* o.519
37 °
0.862 o.171 None detected None detected
" T e s t e d a f t e r a d d i n g M e r t h i o l a t e (see t e x t ) . Biochim. Biophys. Aaa,
55 (1962) 7 6 8 - 7 7 4
772
F. BROWN, B. CARTWRIGHT, D. L. STEWART
tissue-culture cells because cells infected at 37 ° and subsequently washed with E D T A produce as much virus as control cells washed with phosphate-buffered saline. When cells are infected at 37 °, subsequent washing with EDTA does not lead to the recovery of any virus, indicating that in this case the viral antigen is no longer attached to the cell surface. I t was shown in earlier experiments 8 that ether extraction of cells infected at 37 ° led to the recovery of the 7-m# non-infective antigen of the virus but infective virus was not recovered. Similar treatment of cells to which virus had been added at 2 ° led to the recovery of a considerable proportion of the adsorbed infective virus, confirming that disruption of the virus particle had not occurred. In this case, however, the ether disrupts the cells so the result does not provide information regarding the site to which the virus is attached. The complement-fixing activity of cells which have been mixed with virus at 2 ° cannot be measured directly as the test is normally carried out at 37 °. To overcome this difficulty, cells which had been mixed with virus at 2 ° were suspended at 2 ° in lO -8 M Merthiolate (sodium ethylmercurithiosalicylate) for a few hours before the complement-fixation test. This procedure kills cells and so prevents engulfment of virus (see below). Cells treated in this way had a considerable amount of complementfixing antigen on their surface, in contrast to cells infected at 37 ° (Table IV).
E//ect o/ cell debris on virus Debris was prepared from pig-kidney cells b y freezing and thawing suspensions at --7 °0 and + 2 0 ° respectively. Staining these preparations with methylene blue showed that no intact cells remained although a considerable number of nuclei was observed. When the disintegrated cells were added to virus suspensions at 2 ° or at 37 °, considerable loss of infectivity was encountered similar to the losses with intact cells. Extraction of the virus-debris mixtures with phenol resulted in variable recoveries of infective RNA. In most experiments the amount of infective RNA which was recovered was proportional to the residual virus titre, indicating that breakdown of the virus had occurred and the released RNA had been hydrolysed by the ribonuclease in the medium (Table V). In some experiments, however, loss of virus titre was not accompanied by any decrease in the amount of infective RNA which could be recovered. On these occasions the "lost" virus could be recovered by addition of E D T A to the virusdebris mixture. Similar recovery of virus could not be achieved from those mixtures which gave low recoveries of RNA on extraction with phenol. TABLE V INACTIVATION
OF VIRUS
AND
INFECTIVE
RNA
BY
CELL DEBRIS
(log I D so /rnl)
In/eaive R N A (log I D so lrnt)
0.5 5.0
1.5 o.2
5.0 5.2
1.5
Virus
Initial v i r u s Virus + freeze-thawed debris Virus + freeze-thawed debris + 10 -2 M E D T A Virus + g r o u n d debris Virus + g r o u n d debris + IO -s M E D T A
-
6. 3 Biochim.
-
Biophys.
Acta,
-
-
55 (1962) 768-774
773
I N F E C T I O N BY F O O T - A N D - M O U T H D I S E A S E V I R U S
Debris prepared from cells b y grinding in a mortar also inactivated virus but the virus could be recovered b y adding E D T A and the yield of RNA was the same as that obtained from the virus only. Similarly, debris obtained b y grinding freezethawed debris attached virus reversibly but did not disintegrate the virus particle. Presumably grinding cells releases a substance which can disintegrate virus already present on the cells. This substance must be very unstable as the addition of ground debris to virus does not lead to disintegration of virus although attachment occurs. This instability m a y account for the variable results obtained with different preparations of freeze-thawed debris. An interesting observation which m a y be of importance in this connection was made in the phenol extraction of cells and the debris preparations. I n t a c t cells and freeze-thawed debris invariably gave a thin layer of protein at the water-phenol interface whereas ground debris did not, all the protein being at the b o t t o m of the phenol layer.
Location o/ substance in cells which disintegrates virus When virus was added to cells at 2 ° the virus attached but could be removed b y E D T A or ether. Extraction of the cells with phenol yielded as much infective RNA as could be obtained from the virus. If the infected cells were first ground in a mortar at 2 ° in phosphate-buffered saline containing 0.I/~g ribonuclease/ml, taking care t h a t the temperature did not rise, subsequent extraction of the ground cells gave a poor recovery of infective RNA (Table II). The addition of E D T A to these ground cells did not increase the virus titre, in contrast to the increase obtained when E D T A was added to the intact cells. This result suggests that the mechanism for disintegrating virus into its RNA and protein components is situated inside the cell and is released when the cells are ground.
E//ect o/ adding virus to dead cells Cells were killed b y treatment for I h at room temperature with 10 -3 M Merthiolate or 10 -3 M iodoacetate. After this treatment the cells did not metabolize to produce acid in a medium containing glucose nor did they produce any virus after infection. They adsorbed virus, however, to the same extent as untreated cells. The figures in Table VI show typical results obtained with cells killed with Merthiolate. Similar results were obtained with cells killed with iodoacetate. In contrast to normal TABLE DISTRIBUTION
OF INFECTIVE
VI
VIRUS, VIRAL PROTEIN AND KIDNEY CELLS KILLED WITH
Virus
(log ID5o/ml ) Initial virus
6.8
Supernatant
5.0
Cells Cells g r o u n d
with ribonuclease
Cells w a s h e d a t p H 6 . o C e l l s " { - 10 -2 M E D T A
4.5 -o. 5 6.8
R N A AFTER ADDITION MERTHIOLATE Infective R N A (log IDso/ml) I. 7 None detected i .7 1.8 None detected --
Biochim. Biophys. Acta,
OF VIRUS TO PIG-
Complement-fixing activity (ml r [3o complement fixed by z ml antigen) 0.760 o.i8o o.45t) ----
55 ( z 9 6 2 ) 7 6 8 - 7 7 4
774
F. BROWN, B. CARTWRIGHT, D. L. STEWART
cells, however, the a d s o r b e d virus was n o t b r o k e n down to R N A a n d viral p r o t e i n as it could be r e c o v e r e d c o m p l e t e l y b y a d d i t i o n of E D T A to the cells. Also, killed cells to w h i c h virus h a d been a d d e d , a l t h o u g h yielding as m u c h R N A as the i n o c u l a t e d virus, d i d n o t yield a n y R N A if t h e y were first washed with o.15 M acetate, p H 6.0. This i n d i c a t e d t h a t t h e virus was still on t h e outside of t h e cell a n d c o m p l e m e n t - f i x a tion tests confirmed this supposition. A d d i t i o n a l l y , t h e c e l l - v i r u s c o m p l e x y i e l d e d as m u c h infective R N A even a f t e r grinding w i t h ribonuclease, confirming t h a t t h e virus h a d n o t b r o k e n down. DISCUSSION There is l i t t l e i n f o r m a t i o n to show h o w viruses infect a n i m a l cells. A d s o r p t i o n of t h e virus to t h e cell w o u l d a p p e a r to be a necessary first s t e p a n d c u r r e n t ideas on the l a t e r stages of t h e infection process f a v o u r t h e view t h a t t h e infective nucleic acid is released from t h e virus. T h e site at which this t a k e s place, in the case of p i g - k i d n e y tissue-culture cells a n d f o o t - a n d - m o u t h disease virus, a p p e a r s to be within t h e cell. Thus a d s o r p t i o n of t h e virus to cells can occur as efficiently at 2 ° as a t 37 ° b u t t h e virus does n o t e n t e r t h e cell at the lower t e m p e r a t u r e as it can be released b y E D T A w i t h o u t d i s r u p t i n g t h e cell. The a d d i t i o n of s o d i u m d o d e c y l s u l p h a t e to t h e c e l l virus c o m p l e x a t 2 ° does n o t release the virus a l t h o u g h the cells are c o m p l e t e l y d i s r u p t ed. I t w o u l d a p p e a r t h a t t h e r e c e p t o r site on the cell remains a t t a c h e d to t h e virus a n d so p r e v e n t s it infecting o t h e r cells. A t 2 ° no d e t e c t a b l e infective R N A enters t h e cell nor do a n y cells become infected. If t h e v i r u s - c e l l complex is g r o u n d up, however, k e e p i n g t h e t e m p e r a t u r e low, t h e n virus can no longer be recovered b y E D T A ext r a c t i o n , n o r can infective R N A be o b t a i n e d from t h e debris. I t thus a p p e a r s t h a t the m e c h a n i s m for releasing the R N A from t h e virus is n o t on t h e cell surface b u t inside the ceJ1. I t is p r o b a b l y significant t h a t in a few e x p e r i m e n t s in which t h e v i r u s - c e l l c o m p l e x is h e l d at 2 ° for p r o l o n g e d periods, r e c o v e r y of infective R N A is low, indicating t h a t t h e s u b s t a n c e s in t h e cell which are responsible for virus d i s r u p t i o n m a y h a v e leached out of the cell a n d d i s r u p t e d t h e virus on t h e cell surface. Debris prep a r e d from cells b y freeze-thawing can d i s r u p t virus into R N A a n d p r o t e i n even at low t e m p e r a t u r e . I t is likely, therefore, t h a t a l t h o u g h a t t a c h m e n t is efficient at 2 ° , t r a n s p o r t of t h e virus into t h e cell does n o t occur. T h e m e t h o d b y which f o o t - a n d - m o u t h disease virus infects p i g - k i d n e y cells a p p e a r s to be b y an engulfment process which does n o t o p e r a t e at low t e m p e r a t u r e s . This view w o u l d seem to be confirmed b y t h e o b s e r v a t i o n t h a t killed cells, which would n o t be e x p e c t e d to engulf the virus, can a d s o r b t h e virus w i t h o u t releasing its R N A . REFERENCES 1 A. D, HERSHEY AND M. CHASE, J. Gen. Physiol., 36 (1952) 39S. FAZEKASDE ST. GROTH, Nature, 162 (1948) 294. 3 L. HOYLE AND W. FRISCH-NIGGEMEYER, J. Hyg., 53 (1955) 474. 4 L. BOYLE AND N. B. FINTER,
J. Hyg.,
55 (1957) 290.
5 L. C. McLAREN, J. J. HOLLANDAND J. "i'. SYVERTON,J. Exptl, Med., lO9 (1959) 475-
MCLAREN, J. Exptl. Med., lO9 (1959) 487 • W. K. JOKLIK AND J. E. DARNELL, Virology, 13 (1961) 4398 F. BROWN, B. CARTWRIGHTAND D. L. STEWART,Biochim. Biophys. Acta, 47 (1961) 172. 0 H. H. SKINNER, Proc. Roy. Soc. Med., 44 (1951) l°4110 j. B. BROOKSBY, Agr. Research Council Rept., Ser. No. I2 (1952). 11 W. B. MARTINAND W. C*. CHAPMAN, Research Vet. Sci., 2 (1961) 53. 12 S. F. CARTWRIGHT, T. W. F. PAY AND W. M. HENDERSON, J . Gen. Microbiol., 16 (1957) 73 TM 13 R. F. SELLERS, Arch. Virus/orsch., 9 (1959) 621. 14 L. C. MCLAREN, J. J. HOLLANDAND J. T. SYVERTON, J. Exptl. Med., II2 (196o) 581. 6 j . j . HOLLAND AND L. C.
Biochim. Biophys. Aaa, 55 (1962) 768-774