- Email: [email protected]
Effect of ribonuclease of the infectivity of heated arboviruses
Life Sciences Vol . 3, pp . 1039-1045, 1984 . Pergamon Press, Inc. Printed in the United States .
EFFECT OF RIBONUCLEASE ON THE INFECTIYITY OF HEATED ARBOVIFcUSES
Vlsstimil èieyer Institute of Yirology,Czechoslavak Academy of Sciences, Bratislava 9, Czechoslovakia
(Received 1 June 1984)
Changes in infectivity represent the most sensitive indicator of the effect of high temperatures on the biological activity of virus particles (1 ) . The infectivity of virus during the process of thermal inactivation is influenced both by denaturation of surface structural groups of the virus preventing effective adsorption on the host cell, and by the degradation of the viral nucleic acids Structural changes of the capsid occuring during thermal inactivation ere little known particularly in arbovirusesai~e assumed that higher temperatures might injure the viral envelope in such a way that the inner contents of the particle become more accessible to the action of ribonucleeae
( RNA-se
),
u"e therefore studied the effect of RNA-se on the infectivity of different clones of viruses belonging to the tick-borne encephalitis ( TE ) complex ( subgroup B of arboviruses ), and on a clone of Western equine encephalomyelitis ( YrEE ) virus ( subgroup A of arboviruses ), which had been heated for a short time at 50° or 60o C . 1039
RIHONUCLEASE AND HEATED ARBOVIRUSES
1040
Vol . 3, No. 9
Materials and Methods Three times plaque-purified clones of TE-virus strains were used (
Table 1 ),and also a clone designated :~8E-S,
obtained from the prototype strain f-15 of ;'.'EE virus grown in chick embryo cells . The later formed plaques approximately 1 .5 mm in diameter and multiplied in chick embryo cells to a titre of about 1 .8 x 107 plaque-forming units ( PFU ) per ml . Virus materiel in the form of 10 per cent mouse brain suspension ( or in the case of ~lEE-S, the medium consisting of Hanks solution with 10 per cent inactivated horse serum and 0 .5 per cent lactalbumin hydrolysate ) was diluted 1/5 vrith ice-cold phosphate-buffered saline ( BS ), FH 7 .4 ( 0,14 M NaCl, O.G07 M phosphate ) . Aliquots of 2 ml
of the suspension in rubber-atoppered tubes were hea-
ted at 50° or 60° C in a Hôppler type ultrathermostat water bath .Individuil tubes were taken at intervals and cooled in ice-cold water . Aliquots of 0 .6 ml
of each virus
sample were mixed immediately after cooling with the same volume of BS and RNA-se or deoxyribonuclease ( DNA-se ) ( both
30 pg .per ml ,L.Light and Co .,crystalline ), The mixtures were kept 20 minutes at 0°C before tit-
ration . Viruses were titrated in weanling mice ( Dé~in farm ), or in dish cultures of chick embryo cells as previously described ( 2,5 ), Results and Discussion Differences in PFU titres were regularly observed between samples of RNA-ee-treated and untreated SEE virus
RIHONUCLEA3E AND HEATED ARHOVIRUSEB
Vol . 3, No. 9
previously heated at 60°C . 9t the name time no differences were found between DNA-se-treated and untreated virus ( Fig . 1 ) .
WEE/60°C
E
o WEE/60°C+RNA-se WEE/60°C+DNA-se
-2
-3 â -4 ZIZ -5 ô -6 -7 2
4
6 train.
8
10
FI(~. 1 Effect of ribonucleaee ( RNA-ae ) and deozyribonucleeae ( DNA-se ) on the PF[~titre of featern equine encephalomyelitia virus ( iEE ) heated at 60o C . Clones of TE virus
markedly differing from each
other in mouse virulence and in plaque diameter, showed differences both in the degree of thermoinactivation and in response to RNA-ae treatment after 12 minutea~ heating at 50°C . Virus clones Sofin-3 and P-III-E, in which heating caused a drop in PFU titre by about 0.5 and 1 .5 1og10 unite, respectively, also reacted to a lower degree to the action of RNA-ae than clones x~r~a2 and ~r-ßo-3 . In the latter clonaa,heating caused a drop in PFU titre of approzimately 3 .15 1og10 units and subsequent contact with RNA-a~ caused an additional decr~aa~,repeatedly, by 0 .? - 0.9
1041
1042
RIHONUCLEASE AND HEATED ARHOVIitUSES TABLE
Vol . 3, No. 9
1
Strains and Clones of Viruses of the Tick-borne Encephalitis Complex .
Virus strain
Plaque diemeter in mm
Clone
1~ °8 ~5~ ~ ic . sc .
PFU/ml
Hypr
P-III-E
9 .0
8 .5
1.02 x 108
3 - 4
( 48 )
( ~ )
Solin
Solin-3
8.8
7 .5
1 .24 a 108
3 - 4
( 100)
(4 ) 8.2
(1 .0
8 .60 z 105
0 .5-1
8 .2
< l .0
1 .33 z l06
0.5-1
HY-SO-2 **
Hypr
(4 ) HY-~o-3
* = number of intracerebral passages in mice ** = After 49 . intracerebral passage in mice and more than 400 days in persistently infected human amnion cell cultures ( 2,3 ) the virus was plaque purified and passaged 18-times in epithelial hamster kidney cells at 36 .5°C . Both clones were isolated from the 18th passage matQriel . is = intracerebrally sc = aubcutaneously 1og10 unite . IE~1à-as treatment caused no drop in the titre of heated virus suspensions ( Tab .2 ),
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRUSES TABIE 2
Effect of Ribonuclease ( FcNA-ae ) and Deozyribonuclease ( D~1A-se ) on the PFU titre of Clones of Tick-borne Encephalitis Virus heated at 50° for 12 min. log Virus clone
heat
N No
heat + RNA-se
heat + UNA-se
* -0 .5~ * -0 .52
-0" 75 -0 .62
-0.40 -0.24
P-III-E
-1 .65 -1 .44
-1 .95 -1 .79
-1,00 -1 .56
x~r~o-2
-3 .10 -3 .28
-4 .16 -4 .03
-3 .12 -3 .30
x~r-~o-3
-3 .06 -3 .18
--4 .05 -4 .15
-2 .9? -3 .12
Sofia - 3
Nom Plaque assay of original virus
*s Exp .No .I.
N = Plaque assay of treated viruà
** = Exp .No .II .
The treatment of the unheated virus suspensions with pancreatic RNA-as was without effect on their PFU titer. To excluds,that the drop in PFU titre of heated virus after RNA-se treatment was merely an effect of polycationa,we treated the heated virus suspensions with RNA-as inactivated by iodoacetete (4 ) . Aa control esperiu~ent was included the treatment of heated virus suspensions also with lyaozyme ( 30 Rg/ ml , cryata111ne,Armour ) . No significant decrease in the PFU titer was observed except in virus
1043
1044
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRU$ES
suspensions treated with active RNA-se . Under normal conditions the virus envelope is impermeable to free enzymes from the environment, e .g. RNA-se . The present results suggest that gentle heating injures the envelope of the arboviruaea studied to such a degree that their RNA or ribonucleoprotein become accessible to the specific action of RNA-se . Previous results with .infectious RNA iron equine encephalomyelitis virus revealed an absolute dependence of the plating efficiency of this virus on hypertonic solution present during adsorption on chick embryo monolayera (5 ) .
These results suggest
that a further decrease ~n the PFU titre of heated virus suapensions,caused by the action of RNA-se,was due to the decreased number of particles having an intact RNA and capable of adsorbing to and infecting cells in isotonic medium ( containing proteins and other serum components ) . The observed differences in the sensitivity to the action of RNA-se in clones of TE virus heated to 50°C seems to indicate that the degree of thermoreaiatance of the virus clone ahdwa a certain relationship to the atructur~ of the virus envelope . A more pronounced thermolability,elong with a resulting injury of the capsid~ which was manifested in our experiments by e lowered degree of protection of the virus nucleic acid core to the action of RNA-se after heating at 50 °C,indicated a different reaction to some environmental changes in clones of T1's virus which also show lowered virulence . These cloziaa can be thus differentiated from e .g. thermoresiatant virulent clanea of TE viruao The degree of ther-
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRUSES
moresistance seems to be a characteristic marker in
tie
clones of TE virus observed since it remrnined unchan ed both after numerous intracerebral passages in mice ~,d after passages in the yolk sac of 7-day old chick embryos . In several other viruaes,under various conditions, manifestations of lowered resistance of attenuated strains as compared with properties of highly virulent strains have been shown to be suitable for the study of their genetic markers ( 6,7,8 ) . References 1.
S .GARD and O.MAALdöE, The Viruses L ,ed .by F.M.Bur~net and W .1H .Stanley, 8 .359 . Academic Press,New York and London ( 1959 ) .
2,
v .maYER, virology 20, 372 ( 1963 ) .
3e
Q.ldayer, Acta virol . 8, 14 ( 1964 ) .
4.
238, A .M,CRESTFIELD,W .H.STEIN and S.I000EtE, J.Biol .Chem . r 2413 ( 1963 ) "
5.
V .~fAYER and F .SOIC~, Z .Nsturforsch . 16b, 725 ( 1961 ) . r
6.
A .LfOFF, Cold Spring Harbor Symposia 4uant .Bio1 . 27, 159 ( 1962 ),
7.
P.D.000PER, virology 16, 485 ( 1962 ) .
8,
S .HEN(~TSOrT,Z .DINTER and L.PHILIPSOri , Proc .Soc .E~ .Biol . Msd . ( N.Y.) 113, 1019 ( 1963 ) .
1045
EFFECT OF RIBONUCLEASE ON THE INFECTIYITY OF HEATED ARBOVIFcUSES
Vlsstimil èieyer Institute of Yirology,Czechoslavak Academy of Sciences, Bratislava 9, Czechoslovakia
(Received 1 June 1984)
Changes in infectivity represent the most sensitive indicator of the effect of high temperatures on the biological activity of virus particles (1 ) . The infectivity of virus during the process of thermal inactivation is influenced both by denaturation of surface structural groups of the virus preventing effective adsorption on the host cell, and by the degradation of the viral nucleic acids Structural changes of the capsid occuring during thermal inactivation ere little known particularly in arbovirusesai~e assumed that higher temperatures might injure the viral envelope in such a way that the inner contents of the particle become more accessible to the action of ribonucleeae
( RNA-se
),
u"e therefore studied the effect of RNA-se on the infectivity of different clones of viruses belonging to the tick-borne encephalitis ( TE ) complex ( subgroup B of arboviruses ), and on a clone of Western equine encephalomyelitis ( YrEE ) virus ( subgroup A of arboviruses ), which had been heated for a short time at 50° or 60o C . 1039
RIHONUCLEASE AND HEATED ARBOVIRUSES
1040
Vol . 3, No. 9
Materials and Methods Three times plaque-purified clones of TE-virus strains were used (
Table 1 ),and also a clone designated :~8E-S,
obtained from the prototype strain f-15 of ;'.'EE virus grown in chick embryo cells . The later formed plaques approximately 1 .5 mm in diameter and multiplied in chick embryo cells to a titre of about 1 .8 x 107 plaque-forming units ( PFU ) per ml . Virus materiel in the form of 10 per cent mouse brain suspension ( or in the case of ~lEE-S, the medium consisting of Hanks solution with 10 per cent inactivated horse serum and 0 .5 per cent lactalbumin hydrolysate ) was diluted 1/5 vrith ice-cold phosphate-buffered saline ( BS ), FH 7 .4 ( 0,14 M NaCl, O.G07 M phosphate ) . Aliquots of 2 ml
of the suspension in rubber-atoppered tubes were hea-
ted at 50° or 60° C in a Hôppler type ultrathermostat water bath .Individuil tubes were taken at intervals and cooled in ice-cold water . Aliquots of 0 .6 ml
of each virus
sample were mixed immediately after cooling with the same volume of BS and RNA-se or deoxyribonuclease ( DNA-se ) ( both
30 pg .per ml ,L.Light and Co .,crystalline ), The mixtures were kept 20 minutes at 0°C before tit-
ration . Viruses were titrated in weanling mice ( Dé~in farm ), or in dish cultures of chick embryo cells as previously described ( 2,5 ), Results and Discussion Differences in PFU titres were regularly observed between samples of RNA-ee-treated and untreated SEE virus
RIHONUCLEA3E AND HEATED ARHOVIRUSEB
Vol . 3, No. 9
previously heated at 60°C . 9t the name time no differences were found between DNA-se-treated and untreated virus ( Fig . 1 ) .
WEE/60°C
E
o WEE/60°C+RNA-se WEE/60°C+DNA-se
-2
-3 â -4 ZIZ -5 ô -6 -7 2
4
6 train.
8
10
FI(~. 1 Effect of ribonucleaee ( RNA-ae ) and deozyribonucleeae ( DNA-se ) on the PF[~titre of featern equine encephalomyelitia virus ( iEE ) heated at 60o C . Clones of TE virus
markedly differing from each
other in mouse virulence and in plaque diameter, showed differences both in the degree of thermoinactivation and in response to RNA-ae treatment after 12 minutea~ heating at 50°C . Virus clones Sofin-3 and P-III-E, in which heating caused a drop in PFU titre by about 0.5 and 1 .5 1og10 unite, respectively, also reacted to a lower degree to the action of RNA-ae than clones x~r~a2 and ~r-ßo-3 . In the latter clonaa,heating caused a drop in PFU titre of approzimately 3 .15 1og10 units and subsequent contact with RNA-a~ caused an additional decr~aa~,repeatedly, by 0 .? - 0.9
1041
1042
RIHONUCLEASE AND HEATED ARHOVIitUSES TABLE
Vol . 3, No. 9
1
Strains and Clones of Viruses of the Tick-borne Encephalitis Complex .
Virus strain
Plaque diemeter in mm
Clone
1~ °8 ~5~ ~ ic . sc .
PFU/ml
Hypr
P-III-E
9 .0
8 .5
1.02 x 108
3 - 4
( 48 )
( ~ )
Solin
Solin-3
8.8
7 .5
1 .24 a 108
3 - 4
( 100)
(4 ) 8.2
(1 .0
8 .60 z 105
0 .5-1
8 .2
< l .0
1 .33 z l06
0.5-1
HY-SO-2 **
Hypr
(4 ) HY-~o-3
* = number of intracerebral passages in mice ** = After 49 . intracerebral passage in mice and more than 400 days in persistently infected human amnion cell cultures ( 2,3 ) the virus was plaque purified and passaged 18-times in epithelial hamster kidney cells at 36 .5°C . Both clones were isolated from the 18th passage matQriel . is = intracerebrally sc = aubcutaneously 1og10 unite . IE~1à-as treatment caused no drop in the titre of heated virus suspensions ( Tab .2 ),
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRUSES TABIE 2
Effect of Ribonuclease ( FcNA-ae ) and Deozyribonuclease ( D~1A-se ) on the PFU titre of Clones of Tick-borne Encephalitis Virus heated at 50° for 12 min. log Virus clone
heat
N No
heat + RNA-se
heat + UNA-se
* -0 .5~ * -0 .52
-0" 75 -0 .62
-0.40 -0.24
P-III-E
-1 .65 -1 .44
-1 .95 -1 .79
-1,00 -1 .56
x~r~o-2
-3 .10 -3 .28
-4 .16 -4 .03
-3 .12 -3 .30
x~r-~o-3
-3 .06 -3 .18
--4 .05 -4 .15
-2 .9? -3 .12
Sofia - 3
Nom Plaque assay of original virus
*s Exp .No .I.
N = Plaque assay of treated viruà
** = Exp .No .II .
The treatment of the unheated virus suspensions with pancreatic RNA-as was without effect on their PFU titer. To excluds,that the drop in PFU titre of heated virus after RNA-se treatment was merely an effect of polycationa,we treated the heated virus suspensions with RNA-as inactivated by iodoacetete (4 ) . Aa control esperiu~ent was included the treatment of heated virus suspensions also with lyaozyme ( 30 Rg/ ml , cryata111ne,Armour ) . No significant decrease in the PFU titer was observed except in virus
1043
1044
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRU$ES
suspensions treated with active RNA-se . Under normal conditions the virus envelope is impermeable to free enzymes from the environment, e .g. RNA-se . The present results suggest that gentle heating injures the envelope of the arboviruaea studied to such a degree that their RNA or ribonucleoprotein become accessible to the specific action of RNA-se . Previous results with .infectious RNA iron equine encephalomyelitis virus revealed an absolute dependence of the plating efficiency of this virus on hypertonic solution present during adsorption on chick embryo monolayera (5 ) .
These results suggest
that a further decrease ~n the PFU titre of heated virus suapensions,caused by the action of RNA-se,was due to the decreased number of particles having an intact RNA and capable of adsorbing to and infecting cells in isotonic medium ( containing proteins and other serum components ) . The observed differences in the sensitivity to the action of RNA-se in clones of TE virus heated to 50°C seems to indicate that the degree of thermoreaiatance of the virus clone ahdwa a certain relationship to the atructur~ of the virus envelope . A more pronounced thermolability,elong with a resulting injury of the capsid~ which was manifested in our experiments by e lowered degree of protection of the virus nucleic acid core to the action of RNA-se after heating at 50 °C,indicated a different reaction to some environmental changes in clones of T1's virus which also show lowered virulence . These cloziaa can be thus differentiated from e .g. thermoresiatant virulent clanea of TE viruao The degree of ther-
Vol. 3, No . 9
RIHONUCLEASE AND HEATED ARHOVIRUSES
moresistance seems to be a characteristic marker in
tie
clones of TE virus observed since it remrnined unchan ed both after numerous intracerebral passages in mice ~,d after passages in the yolk sac of 7-day old chick embryos . In several other viruaes,under various conditions, manifestations of lowered resistance of attenuated strains as compared with properties of highly virulent strains have been shown to be suitable for the study of their genetic markers ( 6,7,8 ) . References 1.
S .GARD and O.MAALdöE, The Viruses L ,ed .by F.M.Bur~net and W .1H .Stanley, 8 .359 . Academic Press,New York and London ( 1959 ) .
2,
v .maYER, virology 20, 372 ( 1963 ) .
3e
Q.ldayer, Acta virol . 8, 14 ( 1964 ) .
4.
238, A .M,CRESTFIELD,W .H.STEIN and S.I000EtE, J.Biol .Chem . r 2413 ( 1963 ) "
5.
V .~fAYER and F .SOIC~, Z .Nsturforsch . 16b, 725 ( 1961 ) . r
6.
A .LfOFF, Cold Spring Harbor Symposia 4uant .Bio1 . 27, 159 ( 1962 ),
7.
P.D.000PER, virology 16, 485 ( 1962 ) .
8,
S .HEN(~TSOrT,Z .DINTER and L.PHILIPSOri , Proc .Soc .E~ .Biol . Msd . ( N.Y.) 113, 1019 ( 1963 ) .
1045