Journal of Medical Virology 2:127-136 (1978)
Enhanced Parainfluenza I (6/94) Virus Detection in Latently Infected Human Brain Cell Cultures by Treatment With Cytochalasin D and Dimethyl Sulfoxide Zofia Wroblewska, Mary Wellish, and Donald H. Gilden The Multiple Sclerosis Research Center of the Wistar institute, and Department of Neurology of the University of Pennsylvania
The ability of cytochalasin D (CD) and dimethyl sulfoxide (DMSO) t o enhance parainfluenza I (6/94) virus replication was studied in various cell culture systems. Treatment of CVl cells with CD (1 pg/ml) dissolved in DMSO prior t o primary 6/94 virus exposure at 1 Oo - 1O5 multiplicities of infection did not substantially enhance virus replication. However, there was a transient increase in cell associated virus one day after infection of DMSO-treated cultures. CD treatment of cultures of human brain cells latently infected with 6/94 virus (LIHB cells) did not enhance 6 / 9 4 virus detection. Cocultivation of CVl cells with CD-treated LIHB cell cultures, and cocultivation of LIHB cell cultures with CD-treated CV, cells, resulted in the production of both cellassociated and cell-free 6 / 9 4 virus three and five days after cocultivation. N o virus was detected after similar cocultivation of untreated LIHB cell cultures with untreated CVI cells. The usefulness of CD-DMSO treatment in the rescue of virus from 6 / 9 4 LIHB cell cultures appears limited t o a cocultivation system. The use of these techniques to enhance virus rescue from human tissues suspected of harboring latent viral genomes is discussed. Key words: enhanced virus detection, parainfluenza 1 virus, cytochalasin D,dimethyl sulfoxide
I NTR 0DUCTI0N
The cytochalasins, a group of congeneric fungal metabolites, have been shown t o enhance the susceptibility of cells in vitro t o certain RNA virus infections [Deitch et al, 19731 ; cytochalasin D (CD) enhances the infectivity of poliovirus and parainfluenza type
Received for publication February 9, 1977. Address reprint requests t o Zofia Wroblewska, The Multiple Sclerosis Research Center of the Wistar Institute, and Department of Neurology of the University of Pennsylvania, 36 St at Spruce, Philadelphia, PA 19104.
0146-661 5/78/0202-0127$02.00 0 1978 Alan R. Liss, Inc
128
Wroblewska, Wellish, and Gilden
I1 virus in Vero cells [Deitch et al, 19731 . On the other hand. cytochalasin B and D decrease respectively the infectivity of he1 pes simplex virus in human embryonic lung fibroblasts [Dix and Courtney. 19761 and adenovirus in Vero cells [Deitch et al, 19731. We have previously described a model system of latent parainfluenza type I (6/94) virus infection in cultures of cells from the human central nervous system [Wroblewska et al, 19761. In this system, virus was rescued from brain cells only b y cocultivation of infected cells with susceptible indicator cells followed either by subpassaging the mixed cultures or by lysolecithin-induced fusion of brain cells with susceptible indicator cells [Wroblewska et al, 19761. In the continuing search for more direct and sensitive host cell systems for use in detecting and eventually rescuing virus from latently infected cells, the ability of CD t o enhance the detection of parainfluema I was studied in various cell culture systems. METHODS AND MATERIALS Stock Virus
The parainfluenza 1 (6/94) virus was characterized previously [Lewandowski et al, 19741 ; 6/94 is a temperature-sensitive variant that replicates better at 33°C than at 37°C. Stock virus* was propagated in the allantoic cavity of embryonated chicken eggs. and the infectious allantoic fluid was stored at -80°C. Viius titer was 1 X 10' egg infectious doses per ml. Indicator Cells
CV1 (African green monkey kidney) cell cultures, initially seeded with 1 X l o 5 cells, were passaged in T25 plastic flasks. Growth medium consisted of Eagle's Minimum Essential Medium (MEM) supplemented with antibiotics (penicillin 1 0 0 units/ml, and streptomycin 0.05 mg/nil). 0.5% glutaniine, and 10%fetal bovine serum (MEM-FBS). LIHB Cells
The establishment and characterization of' latent 6/94 virus-infected human brain cells (LIHB) has been described [Wroblewska et al, 19761. The virus was cell-associated. No cytopathic effect was observed, and viral antigen was not detectable by hemadsorption (HAD), immunofluorescence (IF), or immunoprecipitation. Infectious virus could only be recovered from LIHB cell cultures by cocultivation followed by subpassaging or by cell fusion. Cytochalasin D Toxicity and Treatment of Indicator Cells
A solution of 1 mg of CD (Aldrich Chemical Company. Milwaukee, Wisconsin) dissolved in 1 ml of dimethyl sulfoxide (DMSO) was diluted to 1 : 10 in deioni7ed water. resulting in a concentration of 100 yg CD per ml and 10% DMSO (stock CD-DMSO). This stock CD-DMSO was passed through a 0.220-y Nalgene filter, aliyuoted and frozen at -20°C. Stock CD-DMSO was further diluted t o various concentrations between 0.1 25 pg/ml and 1 0 pg/ml in Eagle's MEM buffered with 10-20 mM HEPES buffer (HBM). Monolayer cultures, 24 hours old, of both CV1 and human brain cells were incubated in medium containing various concentrations of CD-DMSO, and cytotoxicity was determined *Kindly supplied by Dr David Waters, The Wistar Institute, Philadelphia, Pennsylvania.
Enhanced Parainfluenza I Virus Detection
129
by microscopic observation. Growth medium was removed from indicator cell cultures and replaced b y the highest nontoxic concentrations of CD-DMSO diluted in HBM supplemented with 10% FBS (HBM-FBS). Cells were treated with CD-DMSO for 2 4 hours prior t o inoculation with the 6/94 virus (see below). Control cultures were treated with corresponding dilutions of DMSO in HBM-FBS or with HBM-FBS only. In some experiments, CVI cells were treated with CD-DMSO or DMSO after virus inoculation (see below). Infection of CD-Treated CVI Cell Cultures
Tenfold dilutions of stock virus in MEM-FBS were inoculated at 1 ml per 7'25 flask onto 2-3 CVI cell cultures that had been treated with CD-DMSO or DMSO, or had been left untreated. After virus inoculation, the cultures were incubated for one hour at 33°C. The inoculum was then removed and the cultures were fed with MEM-FBS and incubated for 4-5 days at 33°C in a 5% COz -air atmosphere. Every 2 4 hours for 5 days 0.5 ml of cell culture medium was removed from all experimental and control cultures and stored froien at -80°C. In addition, drug-treated and untreated virus infected cells were grown on glass coverslips and tested at postinfection days 4 and 5 for cell-associated viral antigen by HAD and IF. Cocultivation of Cells (LIHB)
CD-and DMSO-Treated CV1 Cells With Latently Infected Human Brain
LIHB cultures were passaged in T25 Falcon flasks initially seeded with 2-3 X 10' cells in MEM-FBS. Confluent cultures of LIHB were released with a mixture of 0.25% trypsin and 0.1% versene, and the cells were resuspended in MEM-FBS at a concentration of 5 X 10' cells per ml. To each of 2-3 CV, cell cultures (in T25 flasks) that had been treated with CD-DMSO, with DMSO alone, or with HBM-FBS only, I .O ml of the cell suspension was added. After one hour at 33"C, all cultures were fed with HBM-FBS and incubated for 4--5 days at 33°C in a 5% CO,-air atmosphere. In other experiments, the LIHB cell cultures were treated by incubation for 24 hours in CD-DMSO, with DMSO alone, or HBM-FBS only and then cocultivated with untreated CV1 cells as described. Some LIHB cultures pretreated with CD-DMSO and with DMSO were not cocultivated but were subpassaged once. Samples of supernatants from all cultures were collected daily and stored frozen a t -80°C. In addition, cocultivated cultures were monitored for the presence of virus b y HAD and/or I F on post-cocultivation days (PCD) 3-5. Tests for Virus Infectivity
Nine-day-old embryonated hen's eggs were inoculated intraallantoically with 0.2 ml of cell culture supernatants and incubated for five days at 33--35°C. The ability of the allantoic fluid to agglutinate chicken RBC a t 4°C was used a s the indicator of virus presence. Indirect I mmunofluorescence Test
Infected cell cultures were grown on 1 1 X 22 mni glass coverslips in petri dishes, washed in phosphate-buffered saline (PBS), pH 7.2, and then fixed for 5 minutes in acetone. Coverslips were stored at -20°C until used. Cells were stained by the indirect I F staining method [Goldman, 19691 with a 1 :5 dilution of rabbit serum hyperimmune t o 4/94 virus (hemagglutination-inhibition titer 1 :32-1:64), and a 1 : 10 dilution of fluorescein isothiocyanate (F1TC)-conjugated goat anti-rabbit immunoglobulin (IgG) (Baltimore Biologic Laboratory, Cockeysville, Maryland). Controls for monitoring specificity were:
130
Wroblewska, Wellish, and Gilden
1) the absence of fluorescence in uninfected tissue culture cells using the same sera; and 2) a negative control provided by the substitution of normal rabbit serum for the serum hyperimmune to 6/94 virus. Observations were made with a Leitz orthoplan fluorescent microscope illuminated with a mercury HOB 200 bulb, and a Schott BG 12 excitor filter and K510 barrier filter. Hemadsorption
Cell cultures were tested for HAD activity at 4°C with 0.5% guinea pig RBC according to standard methods [Lennette and Schmidt, 19691.
RESULTS CD Toxicity in CV1 and Human Brain Cell Cultures
When the concentration of CD was equal to or less than 1 pg/ml, there was no cytotoxic effect (CTE). CD concentrations greater than 1 pg/ml led to CTE within 24 hours. CTE was characterized by cell shrinkage, intracellular bridges, and the appearance of long, thin cellular processes. Such cultures died within hours after the CTE appeared. Effect of CD and DMSO on 6/94 Virus Infectivity of CVI Cells
CV, cell cultures were treated for 24 hours with CD-DMSO, with DMSO only, or with HBM-FBS and then infected with serial tenfold dilutions of 6/94 virus. The CV, cells were tested for the presence of cell-associated and cell-free virus on post infection day 5 (Table I). At a multiplicity of infection (MOI) of 1, cell-associated virus, as monitored by IIAD, was 20-25% greater than the control in both CD-DMSO-treated and DMSO-treated cultures. In addition, at this MOI, twice as much cell-free virus was detected in DMSOtreated cultures as in CD-DMSO or control cultures. At a lower M01, the level of cellassociated virus rapidly declined in all cultures, and there were no detectable differences, or only slight differences between CD-DMSO-treated or DMSO-treated cultures and control cultures. At the lowest MOI used (.0001), only trace amounts of cell-associated virus were detected and then only in DMSO-treated cultures (Table I). To study the kinetics of virus infection, CD-DMSO-treated and DMSO-treated CV1 cell cultures were inoculated with 6/94 virus at an MOI of 1, and the presence of cellassociated viral antigen in CV, cells was monitored by IF at postinfection days 1 and 4, and by HAD at postinfection day 4. Samples of tissue culture medium were removed daily from postinfection days 1 - 4 to measurc cell-free virus by titration in eggs. In addition, untreated CVI cell cultures were inoculated with 6/94 virus at an MOI of 1, and after virus adsorption then were fed with medium containing CD or DMSO. The presence of cell-associated and cell-free virus was monitored as described above. The results are summarized in Table 11. In all cultures treated with CD-DMSO or with DMSO before infection and in controls, the quantity of cell-free virus was greatest on postinfection day 3. In cultures treated after virus infection, the amount of cell-free virus was greatest on postinfection day 4. Cell-associated virus was 20-30% higher in DMSO-treated cultures than in CD-DMSO-treated cultures, and the greatest amounts of cell-free virus were found in DMSO-treated cultures. The increased amounts of cell-associated and cell-free virus in DMSO-treated cultures were found in cell cultures exposed to DMSO either before or after virus infection (Table 11).
1:2 1:4 < 1:2
HA
~~
70 75 50 ~
3.7 3.7 3.7 ~~
HAD ElDs0/ml
MOI = 1.0
< 1.2 < 1.2
0
HA
HA
0 0 0
40 50 50
MOI
HAD
MOI = 0.1 =
I0 25 10
HAD
0.01
~
0 0 0
HA
~
1 1
Enhanced Parainfluenza I (6/94) Virus Detection in Latently Infected Human Brain Cell Cultures by Treatment With Cytochalasin D and Dimethyl Sulfoxide Zofia Wroblewska, Mary Wellish, and Donald H. Gilden The Multiple Sclerosis Research Center of the Wistar institute, and Department of Neurology of the University of Pennsylvania
The ability of cytochalasin D (CD) and dimethyl sulfoxide (DMSO) t o enhance parainfluenza I (6/94) virus replication was studied in various cell culture systems. Treatment of CVl cells with CD (1 pg/ml) dissolved in DMSO prior t o primary 6/94 virus exposure at 1 Oo - 1O5 multiplicities of infection did not substantially enhance virus replication. However, there was a transient increase in cell associated virus one day after infection of DMSO-treated cultures. CD treatment of cultures of human brain cells latently infected with 6/94 virus (LIHB cells) did not enhance 6 / 9 4 virus detection. Cocultivation of CVl cells with CD-treated LIHB cell cultures, and cocultivation of LIHB cell cultures with CD-treated CV, cells, resulted in the production of both cellassociated and cell-free 6 / 9 4 virus three and five days after cocultivation. N o virus was detected after similar cocultivation of untreated LIHB cell cultures with untreated CVI cells. The usefulness of CD-DMSO treatment in the rescue of virus from 6 / 9 4 LIHB cell cultures appears limited t o a cocultivation system. The use of these techniques to enhance virus rescue from human tissues suspected of harboring latent viral genomes is discussed. Key words: enhanced virus detection, parainfluenza 1 virus, cytochalasin D,dimethyl sulfoxide
I NTR 0DUCTI0N
The cytochalasins, a group of congeneric fungal metabolites, have been shown t o enhance the susceptibility of cells in vitro t o certain RNA virus infections [Deitch et al, 19731 ; cytochalasin D (CD) enhances the infectivity of poliovirus and parainfluenza type
Received for publication February 9, 1977. Address reprint requests t o Zofia Wroblewska, The Multiple Sclerosis Research Center of the Wistar Institute, and Department of Neurology of the University of Pennsylvania, 36 St at Spruce, Philadelphia, PA 19104.
0146-661 5/78/0202-0127$02.00 0 1978 Alan R. Liss, Inc
128
Wroblewska, Wellish, and Gilden
I1 virus in Vero cells [Deitch et al, 19731 . On the other hand. cytochalasin B and D decrease respectively the infectivity of he1 pes simplex virus in human embryonic lung fibroblasts [Dix and Courtney. 19761 and adenovirus in Vero cells [Deitch et al, 19731. We have previously described a model system of latent parainfluenza type I (6/94) virus infection in cultures of cells from the human central nervous system [Wroblewska et al, 19761. In this system, virus was rescued from brain cells only b y cocultivation of infected cells with susceptible indicator cells followed either by subpassaging the mixed cultures or by lysolecithin-induced fusion of brain cells with susceptible indicator cells [Wroblewska et al, 19761. In the continuing search for more direct and sensitive host cell systems for use in detecting and eventually rescuing virus from latently infected cells, the ability of CD t o enhance the detection of parainfluema I was studied in various cell culture systems. METHODS AND MATERIALS Stock Virus
The parainfluenza 1 (6/94) virus was characterized previously [Lewandowski et al, 19741 ; 6/94 is a temperature-sensitive variant that replicates better at 33°C than at 37°C. Stock virus* was propagated in the allantoic cavity of embryonated chicken eggs. and the infectious allantoic fluid was stored at -80°C. Viius titer was 1 X 10' egg infectious doses per ml. Indicator Cells
CV1 (African green monkey kidney) cell cultures, initially seeded with 1 X l o 5 cells, were passaged in T25 plastic flasks. Growth medium consisted of Eagle's Minimum Essential Medium (MEM) supplemented with antibiotics (penicillin 1 0 0 units/ml, and streptomycin 0.05 mg/nil). 0.5% glutaniine, and 10%fetal bovine serum (MEM-FBS). LIHB Cells
The establishment and characterization of' latent 6/94 virus-infected human brain cells (LIHB) has been described [Wroblewska et al, 19761. The virus was cell-associated. No cytopathic effect was observed, and viral antigen was not detectable by hemadsorption (HAD), immunofluorescence (IF), or immunoprecipitation. Infectious virus could only be recovered from LIHB cell cultures by cocultivation followed by subpassaging or by cell fusion. Cytochalasin D Toxicity and Treatment of Indicator Cells
A solution of 1 mg of CD (Aldrich Chemical Company. Milwaukee, Wisconsin) dissolved in 1 ml of dimethyl sulfoxide (DMSO) was diluted to 1 : 10 in deioni7ed water. resulting in a concentration of 100 yg CD per ml and 10% DMSO (stock CD-DMSO). This stock CD-DMSO was passed through a 0.220-y Nalgene filter, aliyuoted and frozen at -20°C. Stock CD-DMSO was further diluted t o various concentrations between 0.1 25 pg/ml and 1 0 pg/ml in Eagle's MEM buffered with 10-20 mM HEPES buffer (HBM). Monolayer cultures, 24 hours old, of both CV1 and human brain cells were incubated in medium containing various concentrations of CD-DMSO, and cytotoxicity was determined *Kindly supplied by Dr David Waters, The Wistar Institute, Philadelphia, Pennsylvania.
Enhanced Parainfluenza I Virus Detection
129
by microscopic observation. Growth medium was removed from indicator cell cultures and replaced b y the highest nontoxic concentrations of CD-DMSO diluted in HBM supplemented with 10% FBS (HBM-FBS). Cells were treated with CD-DMSO for 2 4 hours prior t o inoculation with the 6/94 virus (see below). Control cultures were treated with corresponding dilutions of DMSO in HBM-FBS or with HBM-FBS only. In some experiments, CVI cells were treated with CD-DMSO or DMSO after virus inoculation (see below). Infection of CD-Treated CVI Cell Cultures
Tenfold dilutions of stock virus in MEM-FBS were inoculated at 1 ml per 7'25 flask onto 2-3 CVI cell cultures that had been treated with CD-DMSO or DMSO, or had been left untreated. After virus inoculation, the cultures were incubated for one hour at 33°C. The inoculum was then removed and the cultures were fed with MEM-FBS and incubated for 4-5 days at 33°C in a 5% COz -air atmosphere. Every 2 4 hours for 5 days 0.5 ml of cell culture medium was removed from all experimental and control cultures and stored froien at -80°C. In addition, drug-treated and untreated virus infected cells were grown on glass coverslips and tested at postinfection days 4 and 5 for cell-associated viral antigen by HAD and IF. Cocultivation of Cells (LIHB)
CD-and DMSO-Treated CV1 Cells With Latently Infected Human Brain
LIHB cultures were passaged in T25 Falcon flasks initially seeded with 2-3 X 10' cells in MEM-FBS. Confluent cultures of LIHB were released with a mixture of 0.25% trypsin and 0.1% versene, and the cells were resuspended in MEM-FBS at a concentration of 5 X 10' cells per ml. To each of 2-3 CV, cell cultures (in T25 flasks) that had been treated with CD-DMSO, with DMSO alone, or with HBM-FBS only, I .O ml of the cell suspension was added. After one hour at 33"C, all cultures were fed with HBM-FBS and incubated for 4--5 days at 33°C in a 5% CO,-air atmosphere. In other experiments, the LIHB cell cultures were treated by incubation for 24 hours in CD-DMSO, with DMSO alone, or HBM-FBS only and then cocultivated with untreated CV1 cells as described. Some LIHB cultures pretreated with CD-DMSO and with DMSO were not cocultivated but were subpassaged once. Samples of supernatants from all cultures were collected daily and stored frozen a t -80°C. In addition, cocultivated cultures were monitored for the presence of virus b y HAD and/or I F on post-cocultivation days (PCD) 3-5. Tests for Virus Infectivity
Nine-day-old embryonated hen's eggs were inoculated intraallantoically with 0.2 ml of cell culture supernatants and incubated for five days at 33--35°C. The ability of the allantoic fluid to agglutinate chicken RBC a t 4°C was used a s the indicator of virus presence. Indirect I mmunofluorescence Test
Infected cell cultures were grown on 1 1 X 22 mni glass coverslips in petri dishes, washed in phosphate-buffered saline (PBS), pH 7.2, and then fixed for 5 minutes in acetone. Coverslips were stored at -20°C until used. Cells were stained by the indirect I F staining method [Goldman, 19691 with a 1 :5 dilution of rabbit serum hyperimmune t o 4/94 virus (hemagglutination-inhibition titer 1 :32-1:64), and a 1 : 10 dilution of fluorescein isothiocyanate (F1TC)-conjugated goat anti-rabbit immunoglobulin (IgG) (Baltimore Biologic Laboratory, Cockeysville, Maryland). Controls for monitoring specificity were:
130
Wroblewska, Wellish, and Gilden
1) the absence of fluorescence in uninfected tissue culture cells using the same sera; and 2) a negative control provided by the substitution of normal rabbit serum for the serum hyperimmune to 6/94 virus. Observations were made with a Leitz orthoplan fluorescent microscope illuminated with a mercury HOB 200 bulb, and a Schott BG 12 excitor filter and K510 barrier filter. Hemadsorption
Cell cultures were tested for HAD activity at 4°C with 0.5% guinea pig RBC according to standard methods [Lennette and Schmidt, 19691.
RESULTS CD Toxicity in CV1 and Human Brain Cell Cultures
When the concentration of CD was equal to or less than 1 pg/ml, there was no cytotoxic effect (CTE). CD concentrations greater than 1 pg/ml led to CTE within 24 hours. CTE was characterized by cell shrinkage, intracellular bridges, and the appearance of long, thin cellular processes. Such cultures died within hours after the CTE appeared. Effect of CD and DMSO on 6/94 Virus Infectivity of CVI Cells
CV, cell cultures were treated for 24 hours with CD-DMSO, with DMSO only, or with HBM-FBS and then infected with serial tenfold dilutions of 6/94 virus. The CV, cells were tested for the presence of cell-associated and cell-free virus on post infection day 5 (Table I). At a multiplicity of infection (MOI) of 1, cell-associated virus, as monitored by IIAD, was 20-25% greater than the control in both CD-DMSO-treated and DMSO-treated cultures. In addition, at this MOI, twice as much cell-free virus was detected in DMSOtreated cultures as in CD-DMSO or control cultures. At a lower M01, the level of cellassociated virus rapidly declined in all cultures, and there were no detectable differences, or only slight differences between CD-DMSO-treated or DMSO-treated cultures and control cultures. At the lowest MOI used (.0001), only trace amounts of cell-associated virus were detected and then only in DMSO-treated cultures (Table I). To study the kinetics of virus infection, CD-DMSO-treated and DMSO-treated CV1 cell cultures were inoculated with 6/94 virus at an MOI of 1, and the presence of cellassociated viral antigen in CV, cells was monitored by IF at postinfection days 1 and 4, and by HAD at postinfection day 4. Samples of tissue culture medium were removed daily from postinfection days 1 - 4 to measurc cell-free virus by titration in eggs. In addition, untreated CVI cell cultures were inoculated with 6/94 virus at an MOI of 1, and after virus adsorption then were fed with medium containing CD or DMSO. The presence of cell-associated and cell-free virus was monitored as described above. The results are summarized in Table 11. In all cultures treated with CD-DMSO or with DMSO before infection and in controls, the quantity of cell-free virus was greatest on postinfection day 3. In cultures treated after virus infection, the amount of cell-free virus was greatest on postinfection day 4. Cell-associated virus was 20-30% higher in DMSO-treated cultures than in CD-DMSO-treated cultures, and the greatest amounts of cell-free virus were found in DMSO-treated cultures. The increased amounts of cell-associated and cell-free virus in DMSO-treated cultures were found in cell cultures exposed to DMSO either before or after virus infection (Table 11).
1:2 1:4 < 1:2
HA
~~
70 75 50 ~
3.7 3.7 3.7 ~~
HAD ElDs0/ml
MOI = 1.0
< 1.2 < 1.2
0
HA
HA
0 0 0
40 50 50
MOI
HAD
MOI = 0.1 =
I0 25 10
HAD
0.01
~
0 0 0
HA
~
1 1
