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Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies
Virus Research 67 (2000) 59 – 66 www.elsevier.com/locate/virusres
Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies Christopher D. DeMaula, Kyle R. Bonneau, N. James MacLachlan * Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, Uni6ersity of California, 1126 Haring Hall, Da6is, CA 95616, USA Received 28 October 1999; received in revised form 21 January 2000; accepted 21 January 2000
Abstract Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bluetongue virus; Neutralization epitope; L2 gene; M5 gene
1. Introduction Bluetongue virus (BTV) is the cause of bluetongue, a hemorrhagic disease of sheep and some
* Corresponding author. Tel.: +1-530-7521385; fax: +1530-7548124. E-mail address: [email protected] (N.J. MacLachlan)
species of wild ruminants (Spruell, 1905; MacLachlan, 1994). BTV is a member of the family Reo6iridae and the prototype virus of the genus Orbi6irus (Murphy et al., 1995). There are at least 24 distinct serotypes of BTV, and their distribution throughout the tropical and temperate regions of the world parallels that of Culicoides insect vectors (Gibbs and Greiner, 1994). The BTV particle consists of an icosahedral core formed by five proteins (VP1, VP3, VP4, VP6 and
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VP7) and a diffuse outer coat composed of VP2 and VP5 (Verwoerd et al., 1972; Roy, 1992). The BTV genome consists of ten segments of double stranded RNA, with the L2 and M5 gene segments, respectively, encoding VP2 and VP5 (Mertens et al., 1984). Sheep inoculated with VP2, either isolated from intact particles or generated by in-vitro expression, produce virus neutralizing antibodies and subsequently are resistant to challenge with the homologous BTV serotype (Huismans et al., 1987; Roy et al., 1990). Coexpression of VP2 with VP5 alone, or in combination with core proteins to form double-shelled virus-like particles, enhances the neutralizing antibody response of inoculated sheep (Roy et al., 1990, 1992, 1994). Epitopes on VP2 are responsible for virus neutralization and serotype determination, although VP5 may affect indirectly neutralization through its conformational influence on VP2 (Cowley and Gorman, 1989; Mertens et al., 1989). VP5 also can have a direct role in the neutralization of Orbi6iruses, as VP5-specific monoclonal antibodies (Mabs) were identified recently which neutralized an African horse sickness virus (Martinez-Torrecuadrada et al., 1999). The known neutralizing epitopes of BTV localize to a limited number of apparently interactive domains in VP2 (Gould et al., 1988; Gould and Eaton, 1990; Heidner et al., 1990; Mecham and Jochim, 1990; White and Eaton, 1990; DeMaula et al., 1993; Pierce et al., 1995) and these epitopes sometimes are conserved amongst different BTV serotypes in either a neutralizing or non-neutralizing conformation (Ristow et al., 1988; White and Eaton, 1990; MacLachlan et al., 1992; Rossitto and MacLachlan, 1992). The goal of this study was to further characterize the neutralization epitopes of BTV serotype 10 (BTV10). Specifically, to identify mutations in the L2 and M5 genes of a highly cell culture adapted strain of BTV10 after sequential passage in the presence of different neutralizing Mabs.
specific polyclonal rabbit antiserum and murine Mabs (Mabs 290, 034 and 039) have been described previously (Heidner et al., 1990). Four additional neutralizing Mabs (Mabs 1F12, 2B10, 4H5 and 5E8) were raised against DEMV 034/039, a neutralization resistant viral variant (escape-mutant virus (EMV)) of BTV10, as described previously (Heidner et al., 1990). Mabs to DEMV 034/039 were developed because this EMV was resistant to all neutralizing Mabs in our existing panel (Heidner et al., 1990).
2. Materials and methods
2.3. Neutralization assay
2.1. Antibodies
The various Mabs and viruses were characterized by microneutralization assay (MacLachlan et al., 1992). Briefly, 250 TCID50 of the test virus was
The production and characterization of BTV-
2.2. Viruses The highly cell culture adapted United States prototype strain of BTV10 and the EMVs derived previously from it (EMVs 034, 039 and DEMV 034/039) have been described (Heidner et al., 1990; DeMaula et al., 1993). Six additional EMVs were produced by passaging DEMV 034/039 in the presence of individual neutralizing Mabs, essentially as described previously (Heidner et al., 1990). At least one EMV to each Mab was selected. The EMVs were named after the Mab used for their selection, and for EMVs to Mab 4H5, also by their location in 24-well plates during plaque purification (Table 1). EMVs were plaque-picked at least twice in the presence of the selecting Mab and working stocks of each EMV were prepared by a single passage in BHK-21 cells. A vaccine strain and four field isolates (FIs) of BTV10 also were used to further characterize the Mabs and the neutralization epitopes of BTV. BluVac10 is a modified-live vaccine strain of BTV that was derived from the original 1953 CA8 isolate of BTV10 (Poultry Health Laboratory, CA; McKercher et al., 1970). FI 10B80Y and FI 10O80V were isolated from ruminants in California during 1980 (MacLachlan et al., 1992) and FI 10B90Z and FI 10O90Z were isolated in 1990 (de Mattos et al., 1994).
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added to each well of a microtiter plate containing triplicate twofold dilutions of individual Mabs from mouse ascitic fluids. The plates were incubated for 1 h at 37°C prior to the addition of BHK-21 cells. Neutralization was considered to be at least 50% protection of the monolayer when the virus control wells exhibited 100% cytopathic effect.
2.4. Immune precipitation assay The viral protein binding specificity of Mabs 1F12, 2B10, 4H5, and 5E8 was determined by immune precipitation as described previously for Mabs 034 and 039 (Heidner et al., 1990; Pierce et al., 1995). Briefly, 35S-methionine labeled proteins from DEMV 034/039 infected BHK-21 cells were incubated overnight at 4°C with ascitic fluids containing saturating amounts of each Mab. Immune
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complexes were precipitated with protein-A sepharose (Zymed Laboratories Inc., CA) and prepared for PAGE as described (Heidner et al., 1990).
2.5. Sequencing The L2 and M5 genes of each EMV were sequenced by reverse transcription polymerase chain reaction (RT-PCR) amplification and cycle sequencing of amplicons as reported previously (DeMaula et al., 1993; Bonneau et al., 1999). The synthetic oligonucleotide primers used for amplification and sequencing of the L2 gene have been described (DeMaula et al., 1993). Twenty mer primers (Genosys, TX) used for amplification and sequencing of the M5 gene were designed from the published sequence of the M5 gene of BTV10
Table 1 Neutralization of laboratory and field strains of BTVa Virus
Rabbit antiserum
Monoclonal antibodies Raised to prototype BTV10
Raised to DEMV 034/039
O34
O39
1F12
2B10
4H5
5E8
Escape mutant 6iruses EMV O34 EMV O39 DEMV 034/039 EMV 1F12 EMV 2B10 EMV 4H5-B5 EMV 4H5-C4 EMV 4H5-D5 EMV 5E8
+ + + + + + + + +
− + − − − − − − −
+ − − + − − − − −
− − + − − − − − −
+ − + + − − − − +
− − + − − − − − +
+ + + + + + + + −
Other strains of BTV Prototype BTV10b BluVac 10 FI 10B80Y FI 10O80V FI 10B90Z FI 10O90Z
+ + + + + +
+ + + + + +
+ + + + + +
− − − − − −
+ + + + − −
− − − − − −
− + + w+ − w+
a
+, neutralization (titer\200); −, no neutralization (titerB50); w+, weak neutralization (titer 50–100). The neutralization phenotype of this virus was unchanged after eight serial passages in the presence of non-neutralizing Mab 290. b
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Fig. 1. Immune precipitation of 35S-methionine labeled viral proteins from the neutralization resistant variant virus DEMV 034/039. Lane (1) BTV10 specific rabbit antiserum; (2) Mab 039 (which neutralizes the prototype strain of BTV10); (3) Mab 4H5; (4) Mab 5E8; (5) Mab 1F12; (6) Mab 2B10; (7) Mab 290; (non-neutralizing, VP7-specific).
(Purdy et al., 1986). Sequence data were collected with an ABI 377 automatic sequencer (PE Applied Biosystems, CA) and analyzed with the SEQUENCHER™ program version 3.0 (Gene Codes Corp., MI). The L2 and M5 genes of the various viruses were sequenced in their entirety, and all mutations were confirmed on both the coding and non-coding strands. The amino acid (AA) sequences of VP2 and VP5 were determined and the effects of each mutation on protein secondary structure and hydrophobicity were predicted using the HIBIO MACDNASIS Pro version 3.5 software package (Hitachi Software Engineering Co. Ltd. SanBruno, CA).
3. Results A total of six BTV-specific Mabs were characterized by neutralization assay with a panel of viruses that included both laboratory and field strains of BTV10 (Table 1). Two of these Mabs (Mabs 034 and 039) were raised previously against the prototype strain of BTV10 (Heidner et al., 1990), and four (Mabs 1F12, 2B10, 4H5 and 5E8) were raised against DEMV 034/039. Each
Mab has a distinct pattern of neutralization against the virus panel, and thus a unique epitope binding specificity. Mabs raised against the prototype strain of BTV10 neutralized the prototype, vaccine and field strains of BTV, but generally did not neutralize the EMVs. The neutralization of individual viruses by Mabs raised to DEMV 034/ 039 was also variable. For example, Mab 5E8 neutralized all viruses except the prototype BTV10, EMV 5E8, and FI 10B90Z, whereas Mab 1F12 neutralized only DEMV 034/039 and none of the other viruses evaluated. The protein binding specificity of each Mab was determined by immune precipitation assay. Mabs 4H5 and 5E8 precipitated soluble VP2 from DEMV 034/039 infected BHK-21 cells (Fig. 1). Although Mabs 1F12 and 2B10 neutralized strongly DEMV 034/ 039, they did not precipitate any soluble BTV protein. Similarly, Mab 034 recognizes an epitope on VP2 that is expressed only on intact virions and not individual solubilized BTV proteins (DeMaula et al., 1993). Nine EMVs were evaluated with the Mabs. Three of the EMVs previously were derived from the prototype BTV10, and six additional EMVs were produced by passaging DEMV 034/039 in the presence of individual neutralizing Mabs. The neutralization profiles of individual EMVs were quite variable with the notable exception of EMV 2B10 and the three EMVs raised to Mab 4H5 that had a common pattern of neutralization (Table 1). Interestingly, EMVs 034 and 1F12 also had identical neutralization profiles despite the fact they were selected with different Mabs and that EMV 1F12 was derived from DEMV 034/039 and not directly from the prototype BTV10. The neutralization phenotype of the prototype strain of BTV10 was unchanged after eight serial passages in the presence of a non-neutralizing BTV-specific Mab (Mab 290), confirming that changes in each EMV were the result of the selection process. The L2 and M5 genes of each EMV were sequenced and compared to those of the virus from which they were derived to determine the genetic basis of neutralization (Table 2). Each EMV retained the nucleotide substitutions in the L2 gene of DEMV 034/039. Single nucleotide changes in the L2 genes of EMVs 1F12, 2B10,
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and 4H5 (B5, C4 and D5) likely were responsible for their altered neutralization phenotypes, with each nucleotide change resulting in a single AA substitution in VP2 (Table 2). Interestingly, EMVs 1F12 and 034 had homologous L2 and VP2 sequences, consistent with their identical neutralization phenotypes. The VP2 of EMV 1F12 thus reverted to that of its ancestor EMV 034. EMV 2B10 and the three different EMVs selected with Mab 4H5 also had identical VP2 proteins and neutralization phenotypes. The L2 gene of EMV 5E8 was unchanged from that of DEMV 034/039, whereas nucleotide differences between the M5 genes of these two viruses were likely responsible for the altered neutralization phenotype of EMV 5E8 (Table 2). The M5 genes of the prototype BTV10 and all EMVs other than EMV 5E8 were unchanged and identical to that of the published sequence of the M5 gene of BTV10 (Purdy et al., 1986). All substitutions in VP2 of the EMVs derived from DEMV 034/039 occurred at AA residues 210 or 211, a region identified previously as important to virus neutralization (Gould and Eaton, 1990; DeMaula et al., 1993; Pierce et al., 1995) and possibly virulence (Yamakawa et al., 1994; Bernard et al., 1997). Similarly, the substitution in VP5 of EMV 5E8 was near the amino terminus, a region previously identified as antigenic (WadeEvans et al., 1988; Wang et al., 1995). Each substitution in VP2 of the various EMVs was conservative, but altered the predicted hydropho-
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bicity or secondary structure of the protein. For example, the substitution in EMV 1F12 altered the charge and the hydrophobicity resulting in a predicted VP2 conformation identical to that of EMV 034. The substitution of an arginine for a lysine in VP5 of EMV 5E8 was also conservative, and was predicted to increase the helical conformation in the region of the substitution.
4. Discussion The goal of this study was to further characterize the neutralization determinants of BTV10 by identifying AA substitutions in the VP2 and VP5 proteins of individual EMVs. Previous investigations of the neutralization determinants of BTV have established that multiple conformationally dependent neutralization epitopes exist on VP2, and that these occur in at least two important domains regardless of serotype (Fig. 2). Amino acid substitutions in various EMVs localize the two domains to positions 199–213 (region 1) and 321–346 (region 2) in VP2, although individual AA substitutions also occurred outside these domains (Gould et al., 1988; Gould and Eaton, 1990; DeMaula et al., 1993; Jewell and Mecham, 1994; Pierce et al., 1995). Less definitive studies using peptide blocking or sequence analysis of the L2 gene of different strains of BTV have confirmed the importance of region 1, and identified other regions of VP2 that might contribute to
Table 2 Substitutions in the L2 and M5 genes and VP2 and VP5 proteins of neutralization resistant variant viruses Virus
EMV EMV EMV EMV EMV EMV
a
1F12 2B10 4H5-B5 4H5-C4 4H5-D5 5E8
Nucleotide substitutions (base/position/ substitutiona)
Amino acid substitutions (amino acid/position/ substitutiona)
L2 gene
VP2
G G G G G
678 675 675 675 675
M5 gene
C A A A A
Arg Gly Gly Gly Gly
VP5 211 210 210 210 210
Thr Glu Glu Glu Glu
A 322 G G 323 A
Substitutions in each EMV as compared to the sequence of the parental virus DEMV 034/039.
Lys 98 Arg
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Fig. 2. Proposed neutralization regions (R1 and R2) in the VP2 protein of BTV. *Locations of critical amino acid substitutions in neutralization resistant variant viruses are indicated as reported in this study and by Gould et al. (1988), DeMaula et al. (1993), Jewell and Mecham (1994), Pierce et al. (1995).
neutralization (Hwang and Li, 1993; Bernard et al., 1997). In the present study, substitutions in VP2 of five EMVs that were selected with three different Mabs all were located at either AA residue 210 or 211. The neutralization domain that includes AA residues 210 and 211 clearly is very plastic, as several Mabs raised against DEMV 034/039 recognized neutralizing epitopes that were unique to this virus and absent from the prototype strain of BTV10 from which DEMV 034/039 was derived. We believe this is the first report that a single AA substitution in VP5, without any change in VP2, directly can affect the neutralization of BTV. Studies with reassortant viruses having VP2 and VP5 proteins from different parents previously established that VP5 can influence the neutralization of BTV, likely through its conformational influence on VP2 (Cowley and Gorman, 1989; Mertens et al., 1989). Portions of the amino terminus of VP5 are exposed on the surface of the virion (Yang et al., 1992) and the substitution in VP5 of EMV 5E8 involved strongly polar AAs, which suggests an exterior location where these residues could be hydrated and stabilized by an aqueous environment. Furthermore, a VP5-spe-
cific Mab that neutralized African horse sickness virus bound to an epitope only 6 AAs upstream from the substitution in EMV 5E8 (Martinez-Torrecuadrada et al., 1999). These studies suggest that a region of the amino terminus of VP5, adjacent to AA residue 98, can directly affect the neutralization of BTV. This region could serve as either a Mab binding site or affect virus neutralization through its conformational influence on the neutralization epitopes of VP2. In summary, the neutralization determinants of BTV are highly plastic, interactive, and dependent on the conformation of both VP2 and VP5. They also are restricted to specific regions of the two proteins. Clearly, the considerable plasticity of the neutralization determinants of BTV has significant implications for future development of nonreplicating vaccines.
Acknowledgements The authors gratefully acknowledge Dr Jodi Hedges for computer assistance and Dr Udeni Balasuriya for technical advice. These studies were supported by USDA-NRI competitive grant
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91-37204-6407, funds provided by the Center for Food Animal Health, and the USDA under the Animal Health Act, 1977, Public Law 95-113.
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MacLachlan, N.J., Rossitto, P.V., Heidner, H.W., Iezzi, L.G., Yilma, T.D., DeMaula, C.D., Osburn, B.I., 1992. Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979 – 1981. Vet. Microbiol. 31, 303 – 316. Martinez-Torrecuadrada, J.L., Langeveld, J.P.M., Venteo, A., Sanz, A., Dalsgaard, K., Hamilton, W.D.O., Meloen, R.H., Casal, J.I., 1999. Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus. Virology 257, 449 – 459. McKercher, D.G., Saito, J.K., Singh, K.V., 1970. Serologic evidence of an etiologic role for bluetongue virus in hydranencephaly of calves. J. Am. Vet. Med. Assoc. 156, 1044 – 1047. Mecham, J.O., Jochim, M.M., 1990. Monoclonal antibodies to bluetongue virus define two neutralizing epitopes and a hemagglutinating epitope. Viral Immunol. 3, 161 – 170. Mertens, P.P., Brown, F., Sangar, D.V., 1984. Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135, 207 – 217. Mertens, P.P., Pedley, S., Cowley, J., Burroughs, J.N., Corteyn, A.H., Jeggo, M.H., Jennings, D.M., Gorman, B.M., 1989. Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype. Virology 170, 561 – 565. Murphy, F.A., Fauquet, C.M., Bishop, D.H.L., Ghabrial, S.A., Jarvis, A.W., Martelli, G.P., Mayo, M.A., Summers, M.D., 1995. Virus taxonomy, classification and nomenclature of viruses (Sixth Report of the International Committee on Taxonomy of Viruses). Arch. Virol. Suppl. 10, 412 – 414. Pierce, C.M., Rossitto, P.V., MacLachlan, N.J., 1995. Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17. Virology 209, 263 – 267. Purdy, M.A., Ritter, G.D., Roy, P., 1986. Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. J. Gen. Virol. 67, 957 – 962. Ristow, S., Leendersten, L., Gorham, J., Yilma, T., 1988. Identification of a neutralizing epitope shared by bluetongue virus serotypes 2 and 13. J. Virol. 62, 2502 – 2504. Rossitto, P.V., MacLachlan, N.J., 1992. Neutralizing epitopes of the serotypes of bluetongue virus present in the United States. J. Gen. Virol. 73, 1947 – 1952. Roy, P., 1992. Bluetongue virus proteins. J. Gen. Virol. 73, 3051 – 3064. Roy, P., Urakawa, T., Van Dijk, A.A., Erasmus, B.J., 1990. Recombinant virus vaccine for bluetongue disease in sheep. J. Virol. 64, 1998 – 2003. Roy, P., French, T., Erasmus, B.J., 1992. Protective efficacy of virus-like particles for bluetongue disease. Vaccine 10, 28 – 32. Roy, P., Bishop, D.H.L., LeBlois, H., Erasmus, B.J., 1994. Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection. Vaccine 12, 805 – 811.
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Changes in the outer capsid proteins of bluetongue virus serotype ten that abrogate neutralization by monoclonal antibodies Christopher D. DeMaula, Kyle R. Bonneau, N. James MacLachlan * Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, Uni6ersity of California, 1126 Haring Hall, Da6is, CA 95616, USA Received 28 October 1999; received in revised form 21 January 2000; accepted 21 January 2000
Abstract Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bluetongue virus; Neutralization epitope; L2 gene; M5 gene
1. Introduction Bluetongue virus (BTV) is the cause of bluetongue, a hemorrhagic disease of sheep and some
* Corresponding author. Tel.: +1-530-7521385; fax: +1530-7548124. E-mail address: [email protected] (N.J. MacLachlan)
species of wild ruminants (Spruell, 1905; MacLachlan, 1994). BTV is a member of the family Reo6iridae and the prototype virus of the genus Orbi6irus (Murphy et al., 1995). There are at least 24 distinct serotypes of BTV, and their distribution throughout the tropical and temperate regions of the world parallels that of Culicoides insect vectors (Gibbs and Greiner, 1994). The BTV particle consists of an icosahedral core formed by five proteins (VP1, VP3, VP4, VP6 and
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VP7) and a diffuse outer coat composed of VP2 and VP5 (Verwoerd et al., 1972; Roy, 1992). The BTV genome consists of ten segments of double stranded RNA, with the L2 and M5 gene segments, respectively, encoding VP2 and VP5 (Mertens et al., 1984). Sheep inoculated with VP2, either isolated from intact particles or generated by in-vitro expression, produce virus neutralizing antibodies and subsequently are resistant to challenge with the homologous BTV serotype (Huismans et al., 1987; Roy et al., 1990). Coexpression of VP2 with VP5 alone, or in combination with core proteins to form double-shelled virus-like particles, enhances the neutralizing antibody response of inoculated sheep (Roy et al., 1990, 1992, 1994). Epitopes on VP2 are responsible for virus neutralization and serotype determination, although VP5 may affect indirectly neutralization through its conformational influence on VP2 (Cowley and Gorman, 1989; Mertens et al., 1989). VP5 also can have a direct role in the neutralization of Orbi6iruses, as VP5-specific monoclonal antibodies (Mabs) were identified recently which neutralized an African horse sickness virus (Martinez-Torrecuadrada et al., 1999). The known neutralizing epitopes of BTV localize to a limited number of apparently interactive domains in VP2 (Gould et al., 1988; Gould and Eaton, 1990; Heidner et al., 1990; Mecham and Jochim, 1990; White and Eaton, 1990; DeMaula et al., 1993; Pierce et al., 1995) and these epitopes sometimes are conserved amongst different BTV serotypes in either a neutralizing or non-neutralizing conformation (Ristow et al., 1988; White and Eaton, 1990; MacLachlan et al., 1992; Rossitto and MacLachlan, 1992). The goal of this study was to further characterize the neutralization epitopes of BTV serotype 10 (BTV10). Specifically, to identify mutations in the L2 and M5 genes of a highly cell culture adapted strain of BTV10 after sequential passage in the presence of different neutralizing Mabs.
specific polyclonal rabbit antiserum and murine Mabs (Mabs 290, 034 and 039) have been described previously (Heidner et al., 1990). Four additional neutralizing Mabs (Mabs 1F12, 2B10, 4H5 and 5E8) were raised against DEMV 034/039, a neutralization resistant viral variant (escape-mutant virus (EMV)) of BTV10, as described previously (Heidner et al., 1990). Mabs to DEMV 034/039 were developed because this EMV was resistant to all neutralizing Mabs in our existing panel (Heidner et al., 1990).
2. Materials and methods
2.3. Neutralization assay
2.1. Antibodies
The various Mabs and viruses were characterized by microneutralization assay (MacLachlan et al., 1992). Briefly, 250 TCID50 of the test virus was
The production and characterization of BTV-
2.2. Viruses The highly cell culture adapted United States prototype strain of BTV10 and the EMVs derived previously from it (EMVs 034, 039 and DEMV 034/039) have been described (Heidner et al., 1990; DeMaula et al., 1993). Six additional EMVs were produced by passaging DEMV 034/039 in the presence of individual neutralizing Mabs, essentially as described previously (Heidner et al., 1990). At least one EMV to each Mab was selected. The EMVs were named after the Mab used for their selection, and for EMVs to Mab 4H5, also by their location in 24-well plates during plaque purification (Table 1). EMVs were plaque-picked at least twice in the presence of the selecting Mab and working stocks of each EMV were prepared by a single passage in BHK-21 cells. A vaccine strain and four field isolates (FIs) of BTV10 also were used to further characterize the Mabs and the neutralization epitopes of BTV. BluVac10 is a modified-live vaccine strain of BTV that was derived from the original 1953 CA8 isolate of BTV10 (Poultry Health Laboratory, CA; McKercher et al., 1970). FI 10B80Y and FI 10O80V were isolated from ruminants in California during 1980 (MacLachlan et al., 1992) and FI 10B90Z and FI 10O90Z were isolated in 1990 (de Mattos et al., 1994).
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added to each well of a microtiter plate containing triplicate twofold dilutions of individual Mabs from mouse ascitic fluids. The plates were incubated for 1 h at 37°C prior to the addition of BHK-21 cells. Neutralization was considered to be at least 50% protection of the monolayer when the virus control wells exhibited 100% cytopathic effect.
2.4. Immune precipitation assay The viral protein binding specificity of Mabs 1F12, 2B10, 4H5, and 5E8 was determined by immune precipitation as described previously for Mabs 034 and 039 (Heidner et al., 1990; Pierce et al., 1995). Briefly, 35S-methionine labeled proteins from DEMV 034/039 infected BHK-21 cells were incubated overnight at 4°C with ascitic fluids containing saturating amounts of each Mab. Immune
61
complexes were precipitated with protein-A sepharose (Zymed Laboratories Inc., CA) and prepared for PAGE as described (Heidner et al., 1990).
2.5. Sequencing The L2 and M5 genes of each EMV were sequenced by reverse transcription polymerase chain reaction (RT-PCR) amplification and cycle sequencing of amplicons as reported previously (DeMaula et al., 1993; Bonneau et al., 1999). The synthetic oligonucleotide primers used for amplification and sequencing of the L2 gene have been described (DeMaula et al., 1993). Twenty mer primers (Genosys, TX) used for amplification and sequencing of the M5 gene were designed from the published sequence of the M5 gene of BTV10
Table 1 Neutralization of laboratory and field strains of BTVa Virus
Rabbit antiserum
Monoclonal antibodies Raised to prototype BTV10
Raised to DEMV 034/039
O34
O39
1F12
2B10
4H5
5E8
Escape mutant 6iruses EMV O34 EMV O39 DEMV 034/039 EMV 1F12 EMV 2B10 EMV 4H5-B5 EMV 4H5-C4 EMV 4H5-D5 EMV 5E8
+ + + + + + + + +
− + − − − − − − −
+ − − + − − − − −
− − + − − − − − −
+ − + + − − − − +
− − + − − − − − +
+ + + + + + + + −
Other strains of BTV Prototype BTV10b BluVac 10 FI 10B80Y FI 10O80V FI 10B90Z FI 10O90Z
+ + + + + +
+ + + + + +
+ + + + + +
− − − − − −
+ + + + − −
− − − − − −
− + + w+ − w+
a
+, neutralization (titer\200); −, no neutralization (titerB50); w+, weak neutralization (titer 50–100). The neutralization phenotype of this virus was unchanged after eight serial passages in the presence of non-neutralizing Mab 290. b
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Fig. 1. Immune precipitation of 35S-methionine labeled viral proteins from the neutralization resistant variant virus DEMV 034/039. Lane (1) BTV10 specific rabbit antiserum; (2) Mab 039 (which neutralizes the prototype strain of BTV10); (3) Mab 4H5; (4) Mab 5E8; (5) Mab 1F12; (6) Mab 2B10; (7) Mab 290; (non-neutralizing, VP7-specific).
(Purdy et al., 1986). Sequence data were collected with an ABI 377 automatic sequencer (PE Applied Biosystems, CA) and analyzed with the SEQUENCHER™ program version 3.0 (Gene Codes Corp., MI). The L2 and M5 genes of the various viruses were sequenced in their entirety, and all mutations were confirmed on both the coding and non-coding strands. The amino acid (AA) sequences of VP2 and VP5 were determined and the effects of each mutation on protein secondary structure and hydrophobicity were predicted using the HIBIO MACDNASIS Pro version 3.5 software package (Hitachi Software Engineering Co. Ltd. SanBruno, CA).
3. Results A total of six BTV-specific Mabs were characterized by neutralization assay with a panel of viruses that included both laboratory and field strains of BTV10 (Table 1). Two of these Mabs (Mabs 034 and 039) were raised previously against the prototype strain of BTV10 (Heidner et al., 1990), and four (Mabs 1F12, 2B10, 4H5 and 5E8) were raised against DEMV 034/039. Each
Mab has a distinct pattern of neutralization against the virus panel, and thus a unique epitope binding specificity. Mabs raised against the prototype strain of BTV10 neutralized the prototype, vaccine and field strains of BTV, but generally did not neutralize the EMVs. The neutralization of individual viruses by Mabs raised to DEMV 034/ 039 was also variable. For example, Mab 5E8 neutralized all viruses except the prototype BTV10, EMV 5E8, and FI 10B90Z, whereas Mab 1F12 neutralized only DEMV 034/039 and none of the other viruses evaluated. The protein binding specificity of each Mab was determined by immune precipitation assay. Mabs 4H5 and 5E8 precipitated soluble VP2 from DEMV 034/039 infected BHK-21 cells (Fig. 1). Although Mabs 1F12 and 2B10 neutralized strongly DEMV 034/ 039, they did not precipitate any soluble BTV protein. Similarly, Mab 034 recognizes an epitope on VP2 that is expressed only on intact virions and not individual solubilized BTV proteins (DeMaula et al., 1993). Nine EMVs were evaluated with the Mabs. Three of the EMVs previously were derived from the prototype BTV10, and six additional EMVs were produced by passaging DEMV 034/039 in the presence of individual neutralizing Mabs. The neutralization profiles of individual EMVs were quite variable with the notable exception of EMV 2B10 and the three EMVs raised to Mab 4H5 that had a common pattern of neutralization (Table 1). Interestingly, EMVs 034 and 1F12 also had identical neutralization profiles despite the fact they were selected with different Mabs and that EMV 1F12 was derived from DEMV 034/039 and not directly from the prototype BTV10. The neutralization phenotype of the prototype strain of BTV10 was unchanged after eight serial passages in the presence of a non-neutralizing BTV-specific Mab (Mab 290), confirming that changes in each EMV were the result of the selection process. The L2 and M5 genes of each EMV were sequenced and compared to those of the virus from which they were derived to determine the genetic basis of neutralization (Table 2). Each EMV retained the nucleotide substitutions in the L2 gene of DEMV 034/039. Single nucleotide changes in the L2 genes of EMVs 1F12, 2B10,
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and 4H5 (B5, C4 and D5) likely were responsible for their altered neutralization phenotypes, with each nucleotide change resulting in a single AA substitution in VP2 (Table 2). Interestingly, EMVs 1F12 and 034 had homologous L2 and VP2 sequences, consistent with their identical neutralization phenotypes. The VP2 of EMV 1F12 thus reverted to that of its ancestor EMV 034. EMV 2B10 and the three different EMVs selected with Mab 4H5 also had identical VP2 proteins and neutralization phenotypes. The L2 gene of EMV 5E8 was unchanged from that of DEMV 034/039, whereas nucleotide differences between the M5 genes of these two viruses were likely responsible for the altered neutralization phenotype of EMV 5E8 (Table 2). The M5 genes of the prototype BTV10 and all EMVs other than EMV 5E8 were unchanged and identical to that of the published sequence of the M5 gene of BTV10 (Purdy et al., 1986). All substitutions in VP2 of the EMVs derived from DEMV 034/039 occurred at AA residues 210 or 211, a region identified previously as important to virus neutralization (Gould and Eaton, 1990; DeMaula et al., 1993; Pierce et al., 1995) and possibly virulence (Yamakawa et al., 1994; Bernard et al., 1997). Similarly, the substitution in VP5 of EMV 5E8 was near the amino terminus, a region previously identified as antigenic (WadeEvans et al., 1988; Wang et al., 1995). Each substitution in VP2 of the various EMVs was conservative, but altered the predicted hydropho-
63
bicity or secondary structure of the protein. For example, the substitution in EMV 1F12 altered the charge and the hydrophobicity resulting in a predicted VP2 conformation identical to that of EMV 034. The substitution of an arginine for a lysine in VP5 of EMV 5E8 was also conservative, and was predicted to increase the helical conformation in the region of the substitution.
4. Discussion The goal of this study was to further characterize the neutralization determinants of BTV10 by identifying AA substitutions in the VP2 and VP5 proteins of individual EMVs. Previous investigations of the neutralization determinants of BTV have established that multiple conformationally dependent neutralization epitopes exist on VP2, and that these occur in at least two important domains regardless of serotype (Fig. 2). Amino acid substitutions in various EMVs localize the two domains to positions 199–213 (region 1) and 321–346 (region 2) in VP2, although individual AA substitutions also occurred outside these domains (Gould et al., 1988; Gould and Eaton, 1990; DeMaula et al., 1993; Jewell and Mecham, 1994; Pierce et al., 1995). Less definitive studies using peptide blocking or sequence analysis of the L2 gene of different strains of BTV have confirmed the importance of region 1, and identified other regions of VP2 that might contribute to
Table 2 Substitutions in the L2 and M5 genes and VP2 and VP5 proteins of neutralization resistant variant viruses Virus
EMV EMV EMV EMV EMV EMV
a
1F12 2B10 4H5-B5 4H5-C4 4H5-D5 5E8
Nucleotide substitutions (base/position/ substitutiona)
Amino acid substitutions (amino acid/position/ substitutiona)
L2 gene
VP2
G G G G G
678 675 675 675 675
M5 gene
C A A A A
Arg Gly Gly Gly Gly
VP5 211 210 210 210 210
Thr Glu Glu Glu Glu
A 322 G G 323 A
Substitutions in each EMV as compared to the sequence of the parental virus DEMV 034/039.
Lys 98 Arg
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Fig. 2. Proposed neutralization regions (R1 and R2) in the VP2 protein of BTV. *Locations of critical amino acid substitutions in neutralization resistant variant viruses are indicated as reported in this study and by Gould et al. (1988), DeMaula et al. (1993), Jewell and Mecham (1994), Pierce et al. (1995).
neutralization (Hwang and Li, 1993; Bernard et al., 1997). In the present study, substitutions in VP2 of five EMVs that were selected with three different Mabs all were located at either AA residue 210 or 211. The neutralization domain that includes AA residues 210 and 211 clearly is very plastic, as several Mabs raised against DEMV 034/039 recognized neutralizing epitopes that were unique to this virus and absent from the prototype strain of BTV10 from which DEMV 034/039 was derived. We believe this is the first report that a single AA substitution in VP5, without any change in VP2, directly can affect the neutralization of BTV. Studies with reassortant viruses having VP2 and VP5 proteins from different parents previously established that VP5 can influence the neutralization of BTV, likely through its conformational influence on VP2 (Cowley and Gorman, 1989; Mertens et al., 1989). Portions of the amino terminus of VP5 are exposed on the surface of the virion (Yang et al., 1992) and the substitution in VP5 of EMV 5E8 involved strongly polar AAs, which suggests an exterior location where these residues could be hydrated and stabilized by an aqueous environment. Furthermore, a VP5-spe-
cific Mab that neutralized African horse sickness virus bound to an epitope only 6 AAs upstream from the substitution in EMV 5E8 (Martinez-Torrecuadrada et al., 1999). These studies suggest that a region of the amino terminus of VP5, adjacent to AA residue 98, can directly affect the neutralization of BTV. This region could serve as either a Mab binding site or affect virus neutralization through its conformational influence on the neutralization epitopes of VP2. In summary, the neutralization determinants of BTV are highly plastic, interactive, and dependent on the conformation of both VP2 and VP5. They also are restricted to specific regions of the two proteins. Clearly, the considerable plasticity of the neutralization determinants of BTV has significant implications for future development of nonreplicating vaccines.
Acknowledgements The authors gratefully acknowledge Dr Jodi Hedges for computer assistance and Dr Udeni Balasuriya for technical advice. These studies were supported by USDA-NRI competitive grant
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91-37204-6407, funds provided by the Center for Food Animal Health, and the USDA under the Animal Health Act, 1977, Public Law 95-113.
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