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Observations on the mode of spread of vaccinia virus in HeLa cell monolayer cultures
\TROLOGY
16, (1962)
DISCUSSION AND PRELIMINARY REPORTS Observations on the Mode of Spread Vaccinia Virus in HeLa Cell Monolayer Cultures’
cultures then received maintenance medium containing antivaccinia hyperimmune rabbit strum, t,he other medium containing the same concentration of normal rabbit serum. Two days after infection, primary plaques in the control cultures consist,ed of areas of cell necrosis resulting in the subsequent detachment of degenerated cells from the center of the plaque. On continued incubation for 2&48 hours, numcrous minute secondary plaques developed in such cultures. In cultures containing antiserum, the secondary plaques did not appear. Only primary foci of infection t~~ual in number to primary plaques in control cultures wcrc observed. Since antiserum was only able to inhibit. the formaCon of SecLonclary plaques, n-e conrlutle that it is c:tl):tblc of neutralizing only free extracellular virus. Failure to prevent the formation of primary fori of infections is strongly buggestive of cell-t,o-cell spread of the virus. Foci of infection under antiserum difft~rclcl from ljlaqucs in cont,rols in that they (‘onsieted of slowly tlcveloping mounds of densely clumped ~11s. Central cc%11 nccroris was appreciably delayed and appcart~l only after 2 or more days of continued incubwtion. Tl~us, though antibody is unable to prevent tile formation of foci of infection, it is able to modify their morphology. In another series of experiments the infectivitics of the fluitl and cellular phases of cultures were arsayclti separately ;tt \-wrious tinicl intervals following inoculat,ion with virus. HcLa cell monolayer culturc~ in Leiglit,on tubes (cell density 2000/11~1~1~i were allowed to adsorb 50-200 ~)l:zqurforming units (PFI-) in a short adsorption period of 30 minutes. The culturt~~ were then ~ashcd wi-ith PBS, fed witch Illairlt~~nance nietliuln, and incubated at 37”. Thaw cultures \vcro set aritlc a:: controls, ancl the number of plaques tlevcloping in 2 d:ty:: were countr(l. At Trariou? tiinc> int(~rv:~ls,
of
Various investigators have presented evidence suggesting that herpes simplex (11, herpes B (ej , varicella, and herpes zoster f.3) infections progress by the spread of the virus particles from cell to cell, with little release of virus into the extracellular milieu. The present report describes similar findings with vaccinia virus infection in HcLa cell monolayer cultures. Virus and infected cell suspensions were assayed in HeLa cell cultures (4) by a simplified plaque technique to be reported in detail elsewhere. In brief, l-S-day-old monolayer cultures in Leighton tubes2 (16 x 80 mm) with increased flat surface dimensions (11 x 55 mm) grown in Eagle’s basal medium, supplemented with 0.5% glucose and 105: calf serum, were washed with phosphate-buffered saline (PBS)-pH 7.4. Virus particles or inferted cells were then added to thcsc cultures, suspended in a l&id maintenance medium consisting of 9 parts by volume of a solution of 0.5% glucose, 0.5:4 lactalbumin hydrolyzate, and 0.1% yeast extract in Earle’s balanced salt solution, and 1 part of horse, chicken, or calf serum. Plaques, about 1 mm in diamet,cr, wcrc plainly visible after 2 days’ incubation at, 37” and were counted after the ~11 she&s had been stained with 0.2% neut.ral red solution. TWO types of experiments were designed in order t’o st,udy t,he mode of spread of the virus in the cultures. In one series of experimcnts two sets of monolayer cultures w(arc inoculatrd with 0.2-ml aliquots of an appropriate dilution of virus in PBS. After a 4-llour adsorption period, unadsorbed virus was rtmov-c~tl by washing. One set of ’ Aidctl by :I gr:mt from thr Morton morial Club.
Davis
Me-
01
92
DISCUSSION
AND PRELIMINARY
groups of 4 of the remaining cultures were withdrawn and the medium was assayed for infectivity. The cells from these cultures were then washed, treated with Versene, sedimented, and resuspended in maintenance medium. Aliquots of this suspension, or of appropriate dilutions of it, were then used to determine the number of infected cells. A portion of the concentrated cell suspension was disrupted by a a-minute exposure to sonic oscillation with a 10 kc Raytheon sonic oscillator and likewise assayed for infectivity. Table 1 summarizes the results obtained in a representative experiment. The number of plaques obtained from the disrupted cells within the first 4 hours, during the eclipse phase, was only about 10% of that obtained from the intact cells. By 25 hours, the number of PFU obtained by cell disruption was about twice the number of infected cells. The number of infected cells began to rise between 4 and 6 hours after inoculation of virus, and by 25 hours, each infected cell had given rise to over 200 infected cells. Yet, during this same period, the number of infective particles in the medium remained low, since, at the termination of the experiment at 25 hours, for each PFU detected in the fluid phase of the culture there were over 700 infected cells. Thus, at this TABLE
INFECTIVITYASSAYSOFTHE FLUID AND CELLULAR PHASES OF HELA CELL MONOLAYER CULTURES INFECTED WITH VACCINIA VIRUSQ PFU per culture Time (hours) Medium 0 4 6 11 25
0 0 1 5 63
Intact cells 195 201 375 3200 45,000
time, the number of PFU in the medium was only about 0.1% of that in the cellular phase, whether this was assayed as intact or disrupted cells. This evidence, as well as that obtained from the experiments employing antiserum, suggests that during the early stages of the infection vaccinia virus spreads predominantly from an initially infected cell to immediately adjoining cells. The experimental procedure of assaying infected cells and free virus associated with discrete foci of infections developing within cell sheets is presented as a useful approach to the study of the microepidemiology of viral infections. REFERENCES 1. STOKER, M. G. P., Nature 182, 1525-1526 (1958). 2. BLACK, F. L., and MELNICK, J. L., J. Immunol. 74, 236-242 (1955). 3. WELLER, T. H., Proc. Sot. Exptl. Biol. Med. 83, 340-346 (1953). 4. NISHMI, M., and KELLER, R., Bacterial. Proc. 152, (1961). MOSHE NISHMI ROBERT KELLER Department of Microbiology Michael Reese Hospital and Medical Center Chicago, Illinois Received July 24, 1961
Oxidation
1
Disrupted cells 21 15 340 5625 100,000
a Each culture was allowed to adsorb about 200 PFU of vaccinia virus (plaque counts on 3 control cultures after 2 days’ incubation were 206, 213, 188). At the indicated time intervals groups of 4 cultures were withdrawn and the fluid and cellular phases were assayed for infectivity in HeLa cell cultures by the plaque technique.
REPORTS
in Thermoinactivation
of
Poliovirus
Pretreatment with cystine increases considerably the thermal st,ability of poliovirus as tested by heating at 50”. This thermal stability is assumed to be due to prevention of oxidation by cystine (1). If the action of cystine can be ascribed solely to this mechanism, the degree of thermoresistance obtained by pretreatment with cystine would indicate the extent to which oxidation is responsible for the inactivation of poliovirus at a given temperature. In the present study, the rate of inactivation of poliovirus which hzd been stabilized by cystine and of virus which had not been treated with cystine were determined at different temperatures and at different pH’s. The virus strain used in the experiments was H+, which is a thermolabile strain derived through plaque purification from polio
16, (1962)
DISCUSSION AND PRELIMINARY REPORTS Observations on the Mode of Spread Vaccinia Virus in HeLa Cell Monolayer Cultures’
cultures then received maintenance medium containing antivaccinia hyperimmune rabbit strum, t,he other medium containing the same concentration of normal rabbit serum. Two days after infection, primary plaques in the control cultures consist,ed of areas of cell necrosis resulting in the subsequent detachment of degenerated cells from the center of the plaque. On continued incubation for 2&48 hours, numcrous minute secondary plaques developed in such cultures. In cultures containing antiserum, the secondary plaques did not appear. Only primary foci of infection t~~ual in number to primary plaques in control cultures wcrc observed. Since antiserum was only able to inhibit. the formaCon of SecLonclary plaques, n-e conrlutle that it is c:tl):tblc of neutralizing only free extracellular virus. Failure to prevent the formation of primary fori of infections is strongly buggestive of cell-t,o-cell spread of the virus. Foci of infection under antiserum difft~rclcl from ljlaqucs in cont,rols in that they (‘onsieted of slowly tlcveloping mounds of densely clumped ~11s. Central cc%11 nccroris was appreciably delayed and appcart~l only after 2 or more days of continued incubwtion. Tl~us, though antibody is unable to prevent tile formation of foci of infection, it is able to modify their morphology. In another series of experiments the infectivitics of the fluitl and cellular phases of cultures were arsayclti separately ;tt \-wrious tinicl intervals following inoculat,ion with virus. HcLa cell monolayer culturc~ in Leiglit,on tubes (cell density 2000/11~1~1~i were allowed to adsorb 50-200 ~)l:zqurforming units (PFI-) in a short adsorption period of 30 minutes. The culturt~~ were then ~ashcd wi-ith PBS, fed witch Illairlt~~nance nietliuln, and incubated at 37”. Thaw cultures \vcro set aritlc a:: controls, ancl the number of plaques tlevcloping in 2 d:ty:: were countr(l. At Trariou? tiinc> int(~rv:~ls,
of
Various investigators have presented evidence suggesting that herpes simplex (11, herpes B (ej , varicella, and herpes zoster f.3) infections progress by the spread of the virus particles from cell to cell, with little release of virus into the extracellular milieu. The present report describes similar findings with vaccinia virus infection in HcLa cell monolayer cultures. Virus and infected cell suspensions were assayed in HeLa cell cultures (4) by a simplified plaque technique to be reported in detail elsewhere. In brief, l-S-day-old monolayer cultures in Leighton tubes2 (16 x 80 mm) with increased flat surface dimensions (11 x 55 mm) grown in Eagle’s basal medium, supplemented with 0.5% glucose and 105: calf serum, were washed with phosphate-buffered saline (PBS)-pH 7.4. Virus particles or inferted cells were then added to thcsc cultures, suspended in a l&id maintenance medium consisting of 9 parts by volume of a solution of 0.5% glucose, 0.5:4 lactalbumin hydrolyzate, and 0.1% yeast extract in Earle’s balanced salt solution, and 1 part of horse, chicken, or calf serum. Plaques, about 1 mm in diamet,cr, wcrc plainly visible after 2 days’ incubation at, 37” and were counted after the ~11 she&s had been stained with 0.2% neut.ral red solution. TWO types of experiments were designed in order t’o st,udy t,he mode of spread of the virus in the cultures. In one series of experimcnts two sets of monolayer cultures w(arc inoculatrd with 0.2-ml aliquots of an appropriate dilution of virus in PBS. After a 4-llour adsorption period, unadsorbed virus was rtmov-c~tl by washing. One set of ’ Aidctl by :I gr:mt from thr Morton morial Club.
Davis
Me-
01
92
DISCUSSION
AND PRELIMINARY
groups of 4 of the remaining cultures were withdrawn and the medium was assayed for infectivity. The cells from these cultures were then washed, treated with Versene, sedimented, and resuspended in maintenance medium. Aliquots of this suspension, or of appropriate dilutions of it, were then used to determine the number of infected cells. A portion of the concentrated cell suspension was disrupted by a a-minute exposure to sonic oscillation with a 10 kc Raytheon sonic oscillator and likewise assayed for infectivity. Table 1 summarizes the results obtained in a representative experiment. The number of plaques obtained from the disrupted cells within the first 4 hours, during the eclipse phase, was only about 10% of that obtained from the intact cells. By 25 hours, the number of PFU obtained by cell disruption was about twice the number of infected cells. The number of infected cells began to rise between 4 and 6 hours after inoculation of virus, and by 25 hours, each infected cell had given rise to over 200 infected cells. Yet, during this same period, the number of infective particles in the medium remained low, since, at the termination of the experiment at 25 hours, for each PFU detected in the fluid phase of the culture there were over 700 infected cells. Thus, at this TABLE
INFECTIVITYASSAYSOFTHE FLUID AND CELLULAR PHASES OF HELA CELL MONOLAYER CULTURES INFECTED WITH VACCINIA VIRUSQ PFU per culture Time (hours) Medium 0 4 6 11 25
0 0 1 5 63
Intact cells 195 201 375 3200 45,000
time, the number of PFU in the medium was only about 0.1% of that in the cellular phase, whether this was assayed as intact or disrupted cells. This evidence, as well as that obtained from the experiments employing antiserum, suggests that during the early stages of the infection vaccinia virus spreads predominantly from an initially infected cell to immediately adjoining cells. The experimental procedure of assaying infected cells and free virus associated with discrete foci of infections developing within cell sheets is presented as a useful approach to the study of the microepidemiology of viral infections. REFERENCES 1. STOKER, M. G. P., Nature 182, 1525-1526 (1958). 2. BLACK, F. L., and MELNICK, J. L., J. Immunol. 74, 236-242 (1955). 3. WELLER, T. H., Proc. Sot. Exptl. Biol. Med. 83, 340-346 (1953). 4. NISHMI, M., and KELLER, R., Bacterial. Proc. 152, (1961). MOSHE NISHMI ROBERT KELLER Department of Microbiology Michael Reese Hospital and Medical Center Chicago, Illinois Received July 24, 1961
Oxidation
1
Disrupted cells 21 15 340 5625 100,000
a Each culture was allowed to adsorb about 200 PFU of vaccinia virus (plaque counts on 3 control cultures after 2 days’ incubation were 206, 213, 188). At the indicated time intervals groups of 4 cultures were withdrawn and the fluid and cellular phases were assayed for infectivity in HeLa cell cultures by the plaque technique.
REPORTS
in Thermoinactivation
of
Poliovirus
Pretreatment with cystine increases considerably the thermal st,ability of poliovirus as tested by heating at 50”. This thermal stability is assumed to be due to prevention of oxidation by cystine (1). If the action of cystine can be ascribed solely to this mechanism, the degree of thermoresistance obtained by pretreatment with cystine would indicate the extent to which oxidation is responsible for the inactivation of poliovirus at a given temperature. In the present study, the rate of inactivation of poliovirus which hzd been stabilized by cystine and of virus which had not been treated with cystine were determined at different temperatures and at different pH’s. The virus strain used in the experiments was H+, which is a thermolabile strain derived through plaque purification from polio