IN VITRO Volume14, No. 6, 1978 All rightsreserved 9
AN I M P R O V E D
S Y N C Y T I A I N F E C T I V I T Y ASSAY F O R T H E B O V I N E LEUKEMIA VIRUS'
CLEMENT A. DIGLIO,2CHARLES E. PIPER, 3 ANDJ O R G E F. FERRER Section of Viral Oncology, Comparative Leukemia Studies Unit, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square. Pennsylvania 19348
SUMMARY Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus t BLV~ and BLV-infected lym phocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran IDEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 x 106 viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated bully-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay. Key words: bovine leukemia virus; syncytia infectivity assay; diagnosis of BLV infection. INTRODUCTION Unlike other mammalian C-type leukemia viruses, bovine leukemia virus {BLV~ is capable of inducing rapid syncytia formation in monolayer cell cultures of nontransformed cells from various species (1,2L Based on this observation we have recently developed a simple, specific and quantitative syncytia infectivity assay (SIA) for BLV. The assay can be applied to the detection of BLV-infected lymphocytes, thus providing a method for the direct diagnosis of BLV infection in cattle {3,4). Experiments reported elsewhere {5) have shown that for the diagnosis of BLV infection in cattle the SIA is considerably more sensi-
tire than the electron microscopic detection of virus particles. In this study we have examined several factors that influence the sensitivity of the SIA for cell-free BLV preparations as well as for BLV-infected bovine lymphocytes. MATERIALSAND METHODS
Cells and virus. Bovine embryonic spleen (BESPI cells, used as indicator cells in the SIA, were obtained from a 3-month-old bovine embryo following procedures described earlier (11. Cells were propagated in Eagle's minimal essential medium {MEML supplemented with heat-inactivated ~56~ C, 30 mint 10% fetal bovine serum, penicillin {100 U per mll, and streptomycin il00 'This work was supported in part by USPHS Grant 1PO 1-CA-14193-03, Pennsylvania Department of Agri- /~g per ml). Cells were transferred serially every 5 culture Grant ME4, and USDA Cooperative Agreement to 6 days by dispersing the monolayer with a mix12-14-100-10,675 (45L ture of 0.5% trypsin and 0.06% EDTA. Stocks of ZTo whom requests for reprints should be sent at pres- BESP cells obtained at passages 1, 2 and 3 were ent address: Department of Pathology, New York Medistored in liquid nitrogen with 10% dimethylsulcal College, Valhalla, New York 10595. 3Present address: Hazleton Laboratories America, foxide. The BESP culture was repeatedly tested Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180. for the presence of other viruses and mycoplasma 502
SYNCYTIA INFECTIVITY ASSAY FOR BLV with negative results in all cases. No spontaneous cytopathic changes were observed in the BESP cultures. BLV-infected buffy-coat tBC~ leukocytes were obtained from cows in multiple-case study herd (BF) ~6,7) applying procedures described in a previous study (8). Lymphocytes were isolated by centrifugation 1400 x g, 40 min) of the BC leukocyte suspension ~6 ml) in 4 ml of sodium metrozoate/Fieoll (Lymphoprep, Nyegaard & Co., OsloL The lymphocyte-containing band at the interface was collected and washed twice in M E M before inoculation onto the BESP cells. Lymphocytes were fractionated in discontinuous density gradients made of bovine serum albumin (BSA) (Path-o-cyte 5; Miles Laboratories, Elkhart, Indiana) following the procedure of Kenyon and Piper ~9). The source of BLV was the supernatant fluid of a clone ~cione 1) derived from BLV-infected bat lung (BLV-bat2). This culture was initiated by infecting bat lung cells ITb 1Lu) with BLV and is free of adventitious viruses and mycoplasma. Details on the establishment, maintenance and characteristics of cell line BLV-bat2 have been reported previously. Samples of supernatant fluids from clone 1 were collected on day 4 or 5, filtered through a 0.45-~m Nalgene filter and used as the BLV inoculum. Syncytia infectivity assay. The standard SIA for BLV and BLV-infected leukocytes has been described in detail (4). In brief, 4 x l0 s BESP indicator cells were seeded in 60-ram Falcon tissue-culture dishes (Integrid) and incubated for 24 hr. The monolayer then was treated with diethylamino-ethyl-dextran (DEAL-D) for 30 rain, washed and inoculated with 1 ml of the cellfree BLV inoculum or with 1 ml of the leukocyte suspension. Cultures inoculated with the cell-free BLV preparations were incubated for 2 hr at 37 ~ C. Subsequently, 3 ml of complete medium was added and the cultures were reincubated. Cultures inoculated with leukocytes were incubated for 48 hr after which the monolayer was washed once, refed with complete medium and reincubated. Upon confluency the monolayer was rinsed, fixed with methanol and stained with Giemsa. All samples were tested in duplicate. Cells containing five or more nuclei were scored as syncytia. The infectivity titer of the cell-free inocula was expressed in terms of syncytia forming units (SFU) per ml. To minimize counting errors SFU were determined from duplicate plates inoculated with a dilution of the virus preparation
503
producing 100 to 300 syncytia per dish. The average number of syncytia in these plates then was multiplied by the reciprocal of the dilution. R E S U L T S A N D D ISCUSSION
Previous experiments (unpublished data) suggested that the susceptibility of the indicator BESP cells to the syncytia-inducing effect of BLV decreased with passage. It also was observed that whereas in early passages the BESP cultures consisted predominantly of epithelial-like cells, these were gradually replaced with fibroblastic elements upon further transfer. Experiments then were conducted to determine if the age of the BESP cell cultures influenced their ability to form syncytia upon infection with BLV. For this purpose, two different BLV preparations were titrated on BESP cells from various passage levels. The results (Table 11 dearly show that lowpassage (11 or less) BESP cells are much more susceptible to syncytia induction by BLV than older passage (13 or more) cells. Similar results were obtained when BLV-infected BC lymphocytes were used as the inoculum. These experiments also confirmed earlier observations (3,4j that the number of syncytia on the BESP cell monolayer increases with the number of BC lymphocytes inoculated, and that the optimal BC cell inoculum is 5x l0 s viable lymphocytes per 60-mm Falcon dish. More concentrated BC cell inocula interfered with the growth of the BESP monolayer as well as with its syncytia response. It also was found that there was a close and direct correlation between the viability of the BC cell inocuhun and its syncytia-inducing activity.
TABLE 1 SUSCEPTIBILITY OF B E S P CELLS FROM VARIOUS PASSAGE LEVELS TO THE SYNCYTIA-I NDUCINGACTIVITY OF B L V Virus Inocuhtm a
Stock 1
Passage Level of BESP Cells
AverageSFU/ml (x 10~Ib
10 11.4 13 4.05 19 2.60 38 0.565 Stock 2 3 30.0 7 45.0 27 1.72 47 0.054 a Cell-free supernatant fluid of BLV-bat2 (clone 11. b SFU determined by titration of the virus inoculum (1 mid in serial 10-fold dilutions. Each dilution assayed in duplicate.
504
DIGLIO, PIPER, AND FERRER TABLE 2
REPRODUCIBILIT'tOFTIlES'[ NCYTIAI NFECTI'~rI"t ASSA'ta FORBLV Virus lnoculum
Stock 1 Before freezing After freezing
Date Assayed
AverageSFU / ml
12/12/75 12/30/75 2/20/76 4/12/75 5/12/76
11.4 2.67 2.75 1.52 1.92
qx lOq b
Stock 2 Before freezing After freezing
2/2O/76 138.0 2/25/76 4.10 3/ 3/76 2.50 3/22/76 1.92 4/12/76 1.47 5/12/76 1.82 Using indicator BESP ceils passages 3 to 10. b SFU determined by titration of the virus inoculum (1 ml) in serial 10-fold dilutions. Each dilution assayed in duplicate. In an earlier paper (4) we reported that the syncytia-inducing activity of BLV preparations obtained from the parental BLV-bat2 culture was almost completely lost during storage at - 7 0 ~ C or in liquid nitrogen. This precluded an evaluation of the reproducibility of the SIA for a given preparation in assays performed at different times. Subsequently, the isolation of clone 1 from the BLV-bat~ culture permitted us to obtain much more concentrated BLV preparations from which syncytia-inducing activity could be recovered after freezing. Table 2 shows the results obtained when two BLV preparations from cell line BLVbat2 clone 1 were titrated on the SIA before freezing and at various times after storage at - 7 0 ~ C. Freezing resulted in a marked decrease in the activity of both virus preparations. However, reproducible titers were obtained when aliquots of these preparations were assayed at various periods after freezer storage. Previous studies (3,4) showed that pretreatment of the indicator cells with I)EAE-D markedly enhances their syncytia response to BLV-infected lymphocytes and particularly to cell-free BLV preparations. In these studies we used 25 ~,g per ml of DEAE-D since this was the dose found most effective in enhancing cell penetration of other Ctype viruses (10-13). Experiments were conducted to examine in greater detail the influence of the DEAE-D concentration on the sensitivity of the SIA for BLV and BLV-infected BC cells. The results of these experiments showed, as in previous experiments (4), that the response of the
TABLE 3 EFFECTOFDEAE-D ANDINCUBATIONT [MEOF BLV INOCULUMONTHESYNCYTIARESPONSE OFINI)ICATORBESP CELLS I ncubation Time of Virus Inoeulum a
BESP Cells Pretreated with DEAE-D b
BESP (Jells W ithout I)EAE-D
min
SFU/ml~
SFU/ml ~
Ratio Treated to Untreated
5 1.70x 10*
AN I M P R O V E D
S Y N C Y T I A I N F E C T I V I T Y ASSAY F O R T H E B O V I N E LEUKEMIA VIRUS'
CLEMENT A. DIGLIO,2CHARLES E. PIPER, 3 ANDJ O R G E F. FERRER Section of Viral Oncology, Comparative Leukemia Studies Unit, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square. Pennsylvania 19348
SUMMARY Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus t BLV~ and BLV-infected lym phocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran IDEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 x 106 viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated bully-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay. Key words: bovine leukemia virus; syncytia infectivity assay; diagnosis of BLV infection. INTRODUCTION Unlike other mammalian C-type leukemia viruses, bovine leukemia virus {BLV~ is capable of inducing rapid syncytia formation in monolayer cell cultures of nontransformed cells from various species (1,2L Based on this observation we have recently developed a simple, specific and quantitative syncytia infectivity assay (SIA) for BLV. The assay can be applied to the detection of BLV-infected lymphocytes, thus providing a method for the direct diagnosis of BLV infection in cattle {3,4). Experiments reported elsewhere {5) have shown that for the diagnosis of BLV infection in cattle the SIA is considerably more sensi-
tire than the electron microscopic detection of virus particles. In this study we have examined several factors that influence the sensitivity of the SIA for cell-free BLV preparations as well as for BLV-infected bovine lymphocytes. MATERIALSAND METHODS
Cells and virus. Bovine embryonic spleen (BESPI cells, used as indicator cells in the SIA, were obtained from a 3-month-old bovine embryo following procedures described earlier (11. Cells were propagated in Eagle's minimal essential medium {MEML supplemented with heat-inactivated ~56~ C, 30 mint 10% fetal bovine serum, penicillin {100 U per mll, and streptomycin il00 'This work was supported in part by USPHS Grant 1PO 1-CA-14193-03, Pennsylvania Department of Agri- /~g per ml). Cells were transferred serially every 5 culture Grant ME4, and USDA Cooperative Agreement to 6 days by dispersing the monolayer with a mix12-14-100-10,675 (45L ture of 0.5% trypsin and 0.06% EDTA. Stocks of ZTo whom requests for reprints should be sent at pres- BESP cells obtained at passages 1, 2 and 3 were ent address: Department of Pathology, New York Medistored in liquid nitrogen with 10% dimethylsulcal College, Valhalla, New York 10595. 3Present address: Hazleton Laboratories America, foxide. The BESP culture was repeatedly tested Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180. for the presence of other viruses and mycoplasma 502
SYNCYTIA INFECTIVITY ASSAY FOR BLV with negative results in all cases. No spontaneous cytopathic changes were observed in the BESP cultures. BLV-infected buffy-coat tBC~ leukocytes were obtained from cows in multiple-case study herd (BF) ~6,7) applying procedures described in a previous study (8). Lymphocytes were isolated by centrifugation 1400 x g, 40 min) of the BC leukocyte suspension ~6 ml) in 4 ml of sodium metrozoate/Fieoll (Lymphoprep, Nyegaard & Co., OsloL The lymphocyte-containing band at the interface was collected and washed twice in M E M before inoculation onto the BESP cells. Lymphocytes were fractionated in discontinuous density gradients made of bovine serum albumin (BSA) (Path-o-cyte 5; Miles Laboratories, Elkhart, Indiana) following the procedure of Kenyon and Piper ~9). The source of BLV was the supernatant fluid of a clone ~cione 1) derived from BLV-infected bat lung (BLV-bat2). This culture was initiated by infecting bat lung cells ITb 1Lu) with BLV and is free of adventitious viruses and mycoplasma. Details on the establishment, maintenance and characteristics of cell line BLV-bat2 have been reported previously. Samples of supernatant fluids from clone 1 were collected on day 4 or 5, filtered through a 0.45-~m Nalgene filter and used as the BLV inoculum. Syncytia infectivity assay. The standard SIA for BLV and BLV-infected leukocytes has been described in detail (4). In brief, 4 x l0 s BESP indicator cells were seeded in 60-ram Falcon tissue-culture dishes (Integrid) and incubated for 24 hr. The monolayer then was treated with diethylamino-ethyl-dextran (DEAL-D) for 30 rain, washed and inoculated with 1 ml of the cellfree BLV inoculum or with 1 ml of the leukocyte suspension. Cultures inoculated with the cell-free BLV preparations were incubated for 2 hr at 37 ~ C. Subsequently, 3 ml of complete medium was added and the cultures were reincubated. Cultures inoculated with leukocytes were incubated for 48 hr after which the monolayer was washed once, refed with complete medium and reincubated. Upon confluency the monolayer was rinsed, fixed with methanol and stained with Giemsa. All samples were tested in duplicate. Cells containing five or more nuclei were scored as syncytia. The infectivity titer of the cell-free inocula was expressed in terms of syncytia forming units (SFU) per ml. To minimize counting errors SFU were determined from duplicate plates inoculated with a dilution of the virus preparation
503
producing 100 to 300 syncytia per dish. The average number of syncytia in these plates then was multiplied by the reciprocal of the dilution. R E S U L T S A N D D ISCUSSION
Previous experiments (unpublished data) suggested that the susceptibility of the indicator BESP cells to the syncytia-inducing effect of BLV decreased with passage. It also was observed that whereas in early passages the BESP cultures consisted predominantly of epithelial-like cells, these were gradually replaced with fibroblastic elements upon further transfer. Experiments then were conducted to determine if the age of the BESP cell cultures influenced their ability to form syncytia upon infection with BLV. For this purpose, two different BLV preparations were titrated on BESP cells from various passage levels. The results (Table 11 dearly show that lowpassage (11 or less) BESP cells are much more susceptible to syncytia induction by BLV than older passage (13 or more) cells. Similar results were obtained when BLV-infected BC lymphocytes were used as the inoculum. These experiments also confirmed earlier observations (3,4j that the number of syncytia on the BESP cell monolayer increases with the number of BC lymphocytes inoculated, and that the optimal BC cell inoculum is 5x l0 s viable lymphocytes per 60-mm Falcon dish. More concentrated BC cell inocula interfered with the growth of the BESP monolayer as well as with its syncytia response. It also was found that there was a close and direct correlation between the viability of the BC cell inocuhun and its syncytia-inducing activity.
TABLE 1 SUSCEPTIBILITY OF B E S P CELLS FROM VARIOUS PASSAGE LEVELS TO THE SYNCYTIA-I NDUCINGACTIVITY OF B L V Virus Inocuhtm a
Stock 1
Passage Level of BESP Cells
AverageSFU/ml (x 10~Ib
10 11.4 13 4.05 19 2.60 38 0.565 Stock 2 3 30.0 7 45.0 27 1.72 47 0.054 a Cell-free supernatant fluid of BLV-bat2 (clone 11. b SFU determined by titration of the virus inoculum (1 mid in serial 10-fold dilutions. Each dilution assayed in duplicate.
504
DIGLIO, PIPER, AND FERRER TABLE 2
REPRODUCIBILIT'tOFTIlES'[ NCYTIAI NFECTI'~rI"t ASSA'ta FORBLV Virus lnoculum
Stock 1 Before freezing After freezing
Date Assayed
AverageSFU / ml
12/12/75 12/30/75 2/20/76 4/12/75 5/12/76
11.4 2.67 2.75 1.52 1.92
qx lOq b
Stock 2 Before freezing After freezing
2/2O/76 138.0 2/25/76 4.10 3/ 3/76 2.50 3/22/76 1.92 4/12/76 1.47 5/12/76 1.82 Using indicator BESP ceils passages 3 to 10. b SFU determined by titration of the virus inoculum (1 ml) in serial 10-fold dilutions. Each dilution assayed in duplicate. In an earlier paper (4) we reported that the syncytia-inducing activity of BLV preparations obtained from the parental BLV-bat2 culture was almost completely lost during storage at - 7 0 ~ C or in liquid nitrogen. This precluded an evaluation of the reproducibility of the SIA for a given preparation in assays performed at different times. Subsequently, the isolation of clone 1 from the BLV-bat~ culture permitted us to obtain much more concentrated BLV preparations from which syncytia-inducing activity could be recovered after freezing. Table 2 shows the results obtained when two BLV preparations from cell line BLVbat2 clone 1 were titrated on the SIA before freezing and at various times after storage at - 7 0 ~ C. Freezing resulted in a marked decrease in the activity of both virus preparations. However, reproducible titers were obtained when aliquots of these preparations were assayed at various periods after freezer storage. Previous studies (3,4) showed that pretreatment of the indicator cells with I)EAE-D markedly enhances their syncytia response to BLV-infected lymphocytes and particularly to cell-free BLV preparations. In these studies we used 25 ~,g per ml of DEAE-D since this was the dose found most effective in enhancing cell penetration of other Ctype viruses (10-13). Experiments were conducted to examine in greater detail the influence of the DEAE-D concentration on the sensitivity of the SIA for BLV and BLV-infected BC cells. The results of these experiments showed, as in previous experiments (4), that the response of the
TABLE 3 EFFECTOFDEAE-D ANDINCUBATIONT [MEOF BLV INOCULUMONTHESYNCYTIARESPONSE OFINI)ICATORBESP CELLS I ncubation Time of Virus Inoeulum a
BESP Cells Pretreated with DEAE-D b
BESP (Jells W ithout I)EAE-D
min
SFU/ml~
SFU/ml ~
Ratio Treated to Untreated
5 1.70x 10*
