Microbial Pathogenesis 1990 ; 8 : 2 99-303
Short communication Antigenic topography of bluetongue virus 17 Franziska B . Grieder and Kevin T . Schultz Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, U .S.A .
(Received December 7, 1989 ; accepted January 16, 1990)
Grieder, F . B . (Dept of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, U .S .A .) and K . T . Schultz, Antigenic topography of bluetongue virus 17 . Microbial Pathogenesis 1990 ; 8 : 299-303 . Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization . Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments . Five different epitopes were identified that are involved in neutralization of viral infectivity . Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known . The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed . Key words: bluetongue virus; arbovirus ; neutralization ; epitopes .
Introduction Bluetongue virus (BTV) is a member of the genus Orbivirus, family Reoviridae . Based on serological differences, 24 BTV serotypes have been identified worldwide, and five BTV serotypes are found within the United States .' The virus is composed of 10 segments of double-stranded RNA, seven structural viral proteins, and at least three non-structural proteins . Five viral proteins are found in the inner core of the virus, while the other two viral proteins (P2 and P5) compose the diffuse outer coat of BTV . The major outer coat protein, P2, confers serotype specificity, induces neutralizing antibodies, and is responsible for viral attachment in mammalian cells . Z .3 Recently, the involvement of P5 in BTV serotype determination and virus neutralization has been examined, and evidence derived from experiments performed with reassortants of two different BTV serotypes suggests that conformational determinants of both BTV outer coat proteins interact with neutralizing antibodies to form neutralizing sites .', ' The contribution of humoral versus cellular immunity in protection and recovery of BTV infection, in the ruminant host, has not been completely delineated . However, experiments by Letchworth and Appleton 6 demonstrated that a neutralizing antibody can passively protect lambs from BTV infection . This finding indicates that neutralizing antibodies play an important role in protection from BTV infection . Neutralizing antibody is primarily directed to the major outer coat protein of BTV,' -" but little is known about the topographic relationship of the antigenic determinants of this virus . The objective of the present study was to map neutralizing epitopes of BTV 17 . 0882-4010/90/040299+05 $03 .00/0
© 1990 Academic Press Limited
300
F . B . Grieder and K . T . Schultz
Results and Discussion Two generally accepted methods to map epitopes topographically with mAbs 12 are (1) selection of neutralization-resistant antigenic variant viruses, and (2) competition binding experiments . Neutralization-resistant antigenic variant viruses selected with mAbs can be distinguished from wild type virus by an altered neutralization titer when a panel of mAbs is used in neutralization assays . Hence, the differences in neutralization indicate changes in the antigenic determinants between the viruses . Alternatively, if there is a failure of competition between two mAbs for binding to the virus, this finding indicates that these two antibodies are binding to different epitopes on the virus . In Table 1 the results from the neutralization analysis of seven variant BTV 17 against the panel of mAbs are summarized . One representative neutralization-resistant variant virus generated with each mAb is listed, where V83B2 is the designation given to the variant BTV 17 selected with mAb 83B2 . The relative virus neutralization titer of each mAb is given as the change in neutralization titer (log e ) comparing the neutralization titer of the stock virus with the titer of the variant virus, respectively (e .g . -11 indicates a 2048-fold decrease in the neutralization titer on variant BTV 17 versus stock BTV 17) . Additionally, neutralization assays were performed using different BTV 17 field isolates (provided by Dr J . Stott, University of California-Davis) . A summary of the changes in neutralization titers of four BTV field isolates as compared to the titer with the stock virus is presented in Table 1 . Values were calculated as described above . All BTV field isolates and all neutralization-resistant variant BTV were neutralized by anti-BTV 17 typing serum . There were clear differences in neutralization titers with this panel of eight mAbs to the various viruses, indicating that several distinct epitopes recognized by these mAbs are conformationally dependent ." Five of these mAbs immunoprecipitated the major outer capsid protein, P2, but the other three did not, while none of the eight mAbs bound to any denatured BTV protein . This information is further evidence and strengthens the interpretation of the neutralization results, that several distinct epitopes are involved in BTV neutralization .
Table 1 Results of neutralization assays of variants BTV 17 and BTV 17 field isolates with monoclonal antibodies to BTV BTV
83C8
V83C8a V92F8 V82F5 No .6 No .2 V83B2 V83E8 V91 E2 No . 3 No . 5 V94G10 PPTd EPId
-8e. o -2 0 0 -4` +1 -1 -4` -7` -4` -2 + A
92F8 -5` -12` -9` -5` -5° -5` -7` -8` -10` -5` -5` + B
82F5 -5` -11` -9` -9° -7` -11° -11 _11c -11 , -5` -5` + B
83E8 -2 0 -3 -3 -2 -11` -10` -11 , -3 -2 0 + C,
83B2
91E2
-1` 0 0 0 +2 -11° -5 -11 , -3 -1 +2 + C,
-2 -8` -9` -9` 0 -9` -9` -91 -3 -2 0 C2
94G10
94G9
-4° -3 -9` -9` -2 -1 -3 -4` -8` -4` -5` D
-2 -3 -9` -9` -1 0 -2 -3 -5' -2 -1 E
'Variant BTV 17 = V83C8, V92F8, V82F5, V83B2, V83E8, V91 E2, V94G10 . BTV 17 field isolates = No . 2, No . 3, No . 5, No . 6 . 'Loge change in neutralization titer comparing BTV 17 variants and BTV 17 field isolates with stock BTV 17 (- = decrease, + = increase) . Decrease in neutralization titer is at least 16-fold as compared to stock virus . PPT+ - immunoprecipitation of BTV 17 protein P2 . EPI = epitope designation A-E .
30 1
BTV antigen S
f-
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1 !
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Log green fluorescence Fig . 1 . Competition binding assay using the anti-BTV antibodies 83C8, an IgG, and 91 E2, an IgM . (a) 83C8 in the presence of excess 91 E2; (b) 91 E2 in the presence of excess 83C8 . MFI : control = 18 ; competition assay (a) = 134 ; competition assay (b) = 146 . For details see text .
Two mAbs, 82F5 and 92F8, failed to neutralize any variant BTV 17 . This observation might indicate that the epitope recognized by these two mAb is located in a unique location of the neutralizing antigen . Changes in the antigen near this epitope result in decreased ability of 82F5 and 92F8 to neutralize these viruses . Additionally, despite repeated attempts, no neutralization-resistant variant BTV 17 could be generated with mAb 94G9 . A potential explanation for the lack of successful isolation of variant BTV to mAb 94G9 might be that the antigenic determinant recognized by this mAb is essential for some step in viral infectivity and that the loss of this epitope is lethal for the virus . Competition binding experiments analysed by flow cytometry were performed with this panel of mAbs to evaluate the epitopes further . Representative data from the competition experiment using mAb 83C8 (an IgG) and 91 E2 (an IgM) is given in Fig . 1, where logarithmic green fluorescence on the x-axis is plotted versus cell number on the y-axis . Antibody 83C8 bound to the cell-virus complex in the presence of excess antibody 91 E2 as determined by the fluorescence intensity compared to controls [mean fluorescence intensity (MFI) = 134 versus MFI of each negative control = 18] . When the two mAbs were incubated in reverse sequence the same results were
3 02
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Log green fluorescence Fig . 2 . Competition binding assay using the anti-BTV antibodies 83E8, an IgG, and 91 E2, an IgM . MFI : control = 18 ; competition assay = 70 .
obtained indicating that the observation was not a result of differences in affinities between the two mAbs [Fig . 1(b)] . These results indicate that these two antibodies did not compete with each other for the same binding site and therefore bind to different epitopes . In contrast, there was a significant reduction in the MFI of the cellvirus-83E8 antibody complex when the assay was performed in the presence of excess amounts of antibody 91 E2 (Fig . 2 ; MFI = 70) as compared to the results obtained in the absence of the competing antibody (MFI = 134) . This indicates that these two antibodies (83E8 and 91 E2) did compete for binding to BTV neutralizing site, and that they identify spatially close or overlapping epitopes . Antibodies 82F5, 83B2, 91 E2, 82F8, 94G9 and 94G10 are of the IgM isotype and antibodies 83C8 and 83E8 are IgG, respectively . This enabled us to use this assay for a number of monoclonal antibody competitions to determine their binding sites . Results from this experiment indicated that antibodies 83C8, 82F5, 91 E2, 94G9 and 94G10 bind distinct neutralizing sites . Combining the data from both the virus neutralization experiments with the neutralization-variant viruses and the competition binding assays, the eight mAbs appear to cluster in a minimum of five epitopes that are involved in neutralization . These epitopes were designated A, B, C, D and E . Epitope C appears to be composed of two very close or overlapping epitopes (designated C1 and C2) because of their similar pattern in neutralization and their reduced degree of competition . In summary, five different epitopes on BTV 17 were identified that are involved in neutralization . All five epitopes appear to be conformational, three of these epitopes are clearly associated with P2, and the other two might involve multiple interaction with one or both outer coat proteins . Studies are in progress to characterize these epitopes further. Materials and methods Neutralization resistant variant BTV. To select neutralization resistant variant viruses approximately 10 6 plaque forming units (pfu) of BTV 17 were incubated for 1 h at 4°C with a 10-fold excess of neutralizing mAb as heat-inactivated mouse ascites fluid . The monolayers were overlayed with media containing 0 .5% agarose and half the amount of the original concentration of neutralizing mAb . After an incubation period of 48 h, microscopically visible plaques were
BTV antigen
303
isolated, incubated in subconfluent monolayers of MDBK cells for 48 h or until cell lysis occurred, and virus-containing supernatants were collected and used in standard neutralization assays to ensure that the variant BTV was no longer neutralized by the selecting mAb .
Competition binding experiments . Bovine fibroblast cells (MDBK cells) which are susceptible to BTV infection, were incubated with semi-purified stock BTV 17 at a multiplicity of infection of 100 for 1 h at 4 ° C . Saturating amounts of a first anti-BTV 17 neutralizing mAb (AB1) of the immunoglobulin M isotype (IgM) were incubated for 1 h at 4°C, followed by incubation of saturating amounts of a second anti-BTV 17 neutralizing mAb (AB2) of the immunoglobulin isotype G (IgG) . Cells were then washed with 4°C phosphate buffered saline . Fluorescein isothiocyanate (FITC) conjugated antibody specific for the mouse isotype of the second mAb was then incubated to detect binding of the second mAb in the presence of the first mAb . Controls included (1) MDBK cells and virus incubated with the two anti-BTV mAbs and stained with the FITC conjugated anti-mouse antibody specific for the isotype of the first mAb ; (2) MDBK cells and virus incubated with the two anti-BTV mAb in reverse sequence (i .e . cells+virus+AB2+AB1 +FITC anti-AB1) to exclude the possible competition on the basis of affinity and/or steric hindrance ; (3) MDBK cells and virus incubated with an anti-BTV mAb followed by incubation of a negative control mAb and stained with FITC conjugated antibody specific for the isotype of the negative control mAb ; and (4) MDBK cells and virus incubated with FITC conjugated anti-mouse antibody alone . (3) and (4) are the negative controls .
This work was supported by the US Department of Agriculture grants No . 85-CRSR-2-2632 and No . 87-CRSR-2-3172 . The authors also would like to thank Dr J . Stott, University of California-Davis, for supplying the field isolates of BTV 17 and Linda Black for technical assistance .
References 1 . Knudson DL, Shope RE . Overview of the orbiviruses . In : Barber TL, Jochim MM, eds . Bluetongue and related orbiviruses, New York : Alan R . Liss, 1985; 255-66 . 2 . Huismans H, Van der Walt NT, Cloete M, Erasmus BJ . Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep . Virology 1987 ; 157 : 172-9 . 3 . Verwoerd DW, Els HJ, De Villiers E-M, Huismans H . Structure of the bluetongue virus capsid . J Virol
1972 ;10,783-94 . 4 . Cowley JA, Gorman BM . Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21 . Vet Microbiol 1989; 19 : 37-51 . 5 . Mertens PPL, Pedley S, Cowley J . Analysis of the role of bluetongue virus outer proteins VP2 and VP5 in determination of viral serotypes . Virology 1989 ; 170 : 561-5 . 6 . Letchworth III GL, Appleton JA . Passive protection of mice and sheep against bluetongue virus by neutralizing monoclonal antibody . Infect and Immun 1983 ; 39 : 208-12 . 7 . Appleton J, Letchworth GJ . Monoclonal antibody analysis of serotype restricted and unrestricted bluetongue viral antigenic determinants . Virology 1883 ; 124 : 286-99 . 8 . Huismans H, Erasmus BJ . Identification of the serotype specific antigens of bluetongue virus . Onderstepoort J Vet Res 1981 ; 48 : 51-8 . 9 . Kahlon J, Sugiyama K, Roy P . Molecular basis of bluetongue virus neutralization . J Virol 1983 ; 48 :
627-32 . 10 . Grieder FG, Schultz KT . Conformationally dependent epitopes of bluetongue virus neutralizing antigen . Viral Immunol 1989 ; 2 :17-24 . 11 . Heidner HW, MacLachlan NJ, Fuller FJ, Richards RG, Whetter LE . Bluetongue virus genome remains stable throughout prolonged infection of cattle . J Gen Virol 1988; 69 : 2629-36 . 12 . Gerhard W, Yewdell J, Frankel LE, Webster R . Antigenic structure of influenza virus hemagglutinin defined by hybridoma antibodies . Nature 1981 ; 290 : 713-17 .
Short communication Antigenic topography of bluetongue virus 17 Franziska B . Grieder and Kevin T . Schultz Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, U .S.A .
(Received December 7, 1989 ; accepted January 16, 1990)
Grieder, F . B . (Dept of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, U .S .A .) and K . T . Schultz, Antigenic topography of bluetongue virus 17 . Microbial Pathogenesis 1990 ; 8 : 299-303 . Neutralizing monoclonal antibodies (mAbs) have been produced and used to map the topographical relationship of the surface antigenic determinants of bluetongue virus (BTV) 17 that mediate neutralization . Eight monoclonal antibodies, at least five of which were directed to the major outer coat protein of BTV 17, P2, were studied in neutralization assays using variant BTV 17 and in competition binding experiments . Five different epitopes were identified that are involved in neutralization of viral infectivity . Three of the five epitopes are clearly associated with P2, while the location of the other two epitopes is not known . The potential association of these two epitopes with one or both outer coat proteins of BTV is discussed . Key words: bluetongue virus; arbovirus ; neutralization ; epitopes .
Introduction Bluetongue virus (BTV) is a member of the genus Orbivirus, family Reoviridae . Based on serological differences, 24 BTV serotypes have been identified worldwide, and five BTV serotypes are found within the United States .' The virus is composed of 10 segments of double-stranded RNA, seven structural viral proteins, and at least three non-structural proteins . Five viral proteins are found in the inner core of the virus, while the other two viral proteins (P2 and P5) compose the diffuse outer coat of BTV . The major outer coat protein, P2, confers serotype specificity, induces neutralizing antibodies, and is responsible for viral attachment in mammalian cells . Z .3 Recently, the involvement of P5 in BTV serotype determination and virus neutralization has been examined, and evidence derived from experiments performed with reassortants of two different BTV serotypes suggests that conformational determinants of both BTV outer coat proteins interact with neutralizing antibodies to form neutralizing sites .', ' The contribution of humoral versus cellular immunity in protection and recovery of BTV infection, in the ruminant host, has not been completely delineated . However, experiments by Letchworth and Appleton 6 demonstrated that a neutralizing antibody can passively protect lambs from BTV infection . This finding indicates that neutralizing antibodies play an important role in protection from BTV infection . Neutralizing antibody is primarily directed to the major outer coat protein of BTV,' -" but little is known about the topographic relationship of the antigenic determinants of this virus . The objective of the present study was to map neutralizing epitopes of BTV 17 . 0882-4010/90/040299+05 $03 .00/0
© 1990 Academic Press Limited
300
F . B . Grieder and K . T . Schultz
Results and Discussion Two generally accepted methods to map epitopes topographically with mAbs 12 are (1) selection of neutralization-resistant antigenic variant viruses, and (2) competition binding experiments . Neutralization-resistant antigenic variant viruses selected with mAbs can be distinguished from wild type virus by an altered neutralization titer when a panel of mAbs is used in neutralization assays . Hence, the differences in neutralization indicate changes in the antigenic determinants between the viruses . Alternatively, if there is a failure of competition between two mAbs for binding to the virus, this finding indicates that these two antibodies are binding to different epitopes on the virus . In Table 1 the results from the neutralization analysis of seven variant BTV 17 against the panel of mAbs are summarized . One representative neutralization-resistant variant virus generated with each mAb is listed, where V83B2 is the designation given to the variant BTV 17 selected with mAb 83B2 . The relative virus neutralization titer of each mAb is given as the change in neutralization titer (log e ) comparing the neutralization titer of the stock virus with the titer of the variant virus, respectively (e .g . -11 indicates a 2048-fold decrease in the neutralization titer on variant BTV 17 versus stock BTV 17) . Additionally, neutralization assays were performed using different BTV 17 field isolates (provided by Dr J . Stott, University of California-Davis) . A summary of the changes in neutralization titers of four BTV field isolates as compared to the titer with the stock virus is presented in Table 1 . Values were calculated as described above . All BTV field isolates and all neutralization-resistant variant BTV were neutralized by anti-BTV 17 typing serum . There were clear differences in neutralization titers with this panel of eight mAbs to the various viruses, indicating that several distinct epitopes recognized by these mAbs are conformationally dependent ." Five of these mAbs immunoprecipitated the major outer capsid protein, P2, but the other three did not, while none of the eight mAbs bound to any denatured BTV protein . This information is further evidence and strengthens the interpretation of the neutralization results, that several distinct epitopes are involved in BTV neutralization .
Table 1 Results of neutralization assays of variants BTV 17 and BTV 17 field isolates with monoclonal antibodies to BTV BTV
83C8
V83C8a V92F8 V82F5 No .6 No .2 V83B2 V83E8 V91 E2 No . 3 No . 5 V94G10 PPTd EPId
-8e. o -2 0 0 -4` +1 -1 -4` -7` -4` -2 + A
92F8 -5` -12` -9` -5` -5° -5` -7` -8` -10` -5` -5` + B
82F5 -5` -11` -9` -9° -7` -11° -11 _11c -11 , -5` -5` + B
83E8 -2 0 -3 -3 -2 -11` -10` -11 , -3 -2 0 + C,
83B2
91E2
-1` 0 0 0 +2 -11° -5 -11 , -3 -1 +2 + C,
-2 -8` -9` -9` 0 -9` -9` -91 -3 -2 0 C2
94G10
94G9
-4° -3 -9` -9` -2 -1 -3 -4` -8` -4` -5` D
-2 -3 -9` -9` -1 0 -2 -3 -5' -2 -1 E
'Variant BTV 17 = V83C8, V92F8, V82F5, V83B2, V83E8, V91 E2, V94G10 . BTV 17 field isolates = No . 2, No . 3, No . 5, No . 6 . 'Loge change in neutralization titer comparing BTV 17 variants and BTV 17 field isolates with stock BTV 17 (- = decrease, + = increase) . Decrease in neutralization titer is at least 16-fold as compared to stock virus . PPT+ - immunoprecipitation of BTV 17 protein P2 . EPI = epitope designation A-E .
30 1
BTV antigen S
f-
-
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(0) t f ff 91E2 +83C8 i
1 !
t s
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t t
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1 S
(b)
f
5f f
fS 83C8 + 91E2
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Log green fluorescence Fig . 1 . Competition binding assay using the anti-BTV antibodies 83C8, an IgG, and 91 E2, an IgM . (a) 83C8 in the presence of excess 91 E2; (b) 91 E2 in the presence of excess 83C8 . MFI : control = 18 ; competition assay (a) = 134 ; competition assay (b) = 146 . For details see text .
Two mAbs, 82F5 and 92F8, failed to neutralize any variant BTV 17 . This observation might indicate that the epitope recognized by these two mAb is located in a unique location of the neutralizing antigen . Changes in the antigen near this epitope result in decreased ability of 82F5 and 92F8 to neutralize these viruses . Additionally, despite repeated attempts, no neutralization-resistant variant BTV 17 could be generated with mAb 94G9 . A potential explanation for the lack of successful isolation of variant BTV to mAb 94G9 might be that the antigenic determinant recognized by this mAb is essential for some step in viral infectivity and that the loss of this epitope is lethal for the virus . Competition binding experiments analysed by flow cytometry were performed with this panel of mAbs to evaluate the epitopes further . Representative data from the competition experiment using mAb 83C8 (an IgG) and 91 E2 (an IgM) is given in Fig . 1, where logarithmic green fluorescence on the x-axis is plotted versus cell number on the y-axis . Antibody 83C8 bound to the cell-virus complex in the presence of excess antibody 91 E2 as determined by the fluorescence intensity compared to controls [mean fluorescence intensity (MFI) = 134 versus MFI of each negative control = 18] . When the two mAbs were incubated in reverse sequence the same results were
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Log green fluorescence Fig . 2 . Competition binding assay using the anti-BTV antibodies 83E8, an IgG, and 91 E2, an IgM . MFI : control = 18 ; competition assay = 70 .
obtained indicating that the observation was not a result of differences in affinities between the two mAbs [Fig . 1(b)] . These results indicate that these two antibodies did not compete with each other for the same binding site and therefore bind to different epitopes . In contrast, there was a significant reduction in the MFI of the cellvirus-83E8 antibody complex when the assay was performed in the presence of excess amounts of antibody 91 E2 (Fig . 2 ; MFI = 70) as compared to the results obtained in the absence of the competing antibody (MFI = 134) . This indicates that these two antibodies (83E8 and 91 E2) did compete for binding to BTV neutralizing site, and that they identify spatially close or overlapping epitopes . Antibodies 82F5, 83B2, 91 E2, 82F8, 94G9 and 94G10 are of the IgM isotype and antibodies 83C8 and 83E8 are IgG, respectively . This enabled us to use this assay for a number of monoclonal antibody competitions to determine their binding sites . Results from this experiment indicated that antibodies 83C8, 82F5, 91 E2, 94G9 and 94G10 bind distinct neutralizing sites . Combining the data from both the virus neutralization experiments with the neutralization-variant viruses and the competition binding assays, the eight mAbs appear to cluster in a minimum of five epitopes that are involved in neutralization . These epitopes were designated A, B, C, D and E . Epitope C appears to be composed of two very close or overlapping epitopes (designated C1 and C2) because of their similar pattern in neutralization and their reduced degree of competition . In summary, five different epitopes on BTV 17 were identified that are involved in neutralization . All five epitopes appear to be conformational, three of these epitopes are clearly associated with P2, and the other two might involve multiple interaction with one or both outer coat proteins . Studies are in progress to characterize these epitopes further. Materials and methods Neutralization resistant variant BTV. To select neutralization resistant variant viruses approximately 10 6 plaque forming units (pfu) of BTV 17 were incubated for 1 h at 4°C with a 10-fold excess of neutralizing mAb as heat-inactivated mouse ascites fluid . The monolayers were overlayed with media containing 0 .5% agarose and half the amount of the original concentration of neutralizing mAb . After an incubation period of 48 h, microscopically visible plaques were
BTV antigen
303
isolated, incubated in subconfluent monolayers of MDBK cells for 48 h or until cell lysis occurred, and virus-containing supernatants were collected and used in standard neutralization assays to ensure that the variant BTV was no longer neutralized by the selecting mAb .
Competition binding experiments . Bovine fibroblast cells (MDBK cells) which are susceptible to BTV infection, were incubated with semi-purified stock BTV 17 at a multiplicity of infection of 100 for 1 h at 4 ° C . Saturating amounts of a first anti-BTV 17 neutralizing mAb (AB1) of the immunoglobulin M isotype (IgM) were incubated for 1 h at 4°C, followed by incubation of saturating amounts of a second anti-BTV 17 neutralizing mAb (AB2) of the immunoglobulin isotype G (IgG) . Cells were then washed with 4°C phosphate buffered saline . Fluorescein isothiocyanate (FITC) conjugated antibody specific for the mouse isotype of the second mAb was then incubated to detect binding of the second mAb in the presence of the first mAb . Controls included (1) MDBK cells and virus incubated with the two anti-BTV mAbs and stained with the FITC conjugated anti-mouse antibody specific for the isotype of the first mAb ; (2) MDBK cells and virus incubated with the two anti-BTV mAb in reverse sequence (i .e . cells+virus+AB2+AB1 +FITC anti-AB1) to exclude the possible competition on the basis of affinity and/or steric hindrance ; (3) MDBK cells and virus incubated with an anti-BTV mAb followed by incubation of a negative control mAb and stained with FITC conjugated antibody specific for the isotype of the negative control mAb ; and (4) MDBK cells and virus incubated with FITC conjugated anti-mouse antibody alone . (3) and (4) are the negative controls .
This work was supported by the US Department of Agriculture grants No . 85-CRSR-2-2632 and No . 87-CRSR-2-3172 . The authors also would like to thank Dr J . Stott, University of California-Davis, for supplying the field isolates of BTV 17 and Linda Black for technical assistance .
References 1 . Knudson DL, Shope RE . Overview of the orbiviruses . In : Barber TL, Jochim MM, eds . Bluetongue and related orbiviruses, New York : Alan R . Liss, 1985; 255-66 . 2 . Huismans H, Van der Walt NT, Cloete M, Erasmus BJ . Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep . Virology 1987 ; 157 : 172-9 . 3 . Verwoerd DW, Els HJ, De Villiers E-M, Huismans H . Structure of the bluetongue virus capsid . J Virol
1972 ;10,783-94 . 4 . Cowley JA, Gorman BM . Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21 . Vet Microbiol 1989; 19 : 37-51 . 5 . Mertens PPL, Pedley S, Cowley J . Analysis of the role of bluetongue virus outer proteins VP2 and VP5 in determination of viral serotypes . Virology 1989 ; 170 : 561-5 . 6 . Letchworth III GL, Appleton JA . Passive protection of mice and sheep against bluetongue virus by neutralizing monoclonal antibody . Infect and Immun 1983 ; 39 : 208-12 . 7 . Appleton J, Letchworth GJ . Monoclonal antibody analysis of serotype restricted and unrestricted bluetongue viral antigenic determinants . Virology 1883 ; 124 : 286-99 . 8 . Huismans H, Erasmus BJ . Identification of the serotype specific antigens of bluetongue virus . Onderstepoort J Vet Res 1981 ; 48 : 51-8 . 9 . Kahlon J, Sugiyama K, Roy P . Molecular basis of bluetongue virus neutralization . J Virol 1983 ; 48 :
627-32 . 10 . Grieder FG, Schultz KT . Conformationally dependent epitopes of bluetongue virus neutralizing antigen . Viral Immunol 1989 ; 2 :17-24 . 11 . Heidner HW, MacLachlan NJ, Fuller FJ, Richards RG, Whetter LE . Bluetongue virus genome remains stable throughout prolonged infection of cattle . J Gen Virol 1988; 69 : 2629-36 . 12 . Gerhard W, Yewdell J, Frankel LE, Webster R . Antigenic structure of influenza virus hemagglutinin defined by hybridoma antibodies . Nature 1981 ; 290 : 713-17 .
