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Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection
Veterinary Microbiology, 16 (1988) 231-241 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
231
Temporal Development of Bluetongue Virus Protein-specific Antibody in Sheep following Natural Infection* M.A. ADKISON 1, J.L. STOTT' and B.I. OSBURN 2
'Department of Microbiology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) 2Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted for publication 6 October 1987)
ABSTRACT Adkison, M.A., Stott, J.L. and Osburn, B.I., 1988. Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection. Vet. Microbiol., 16: 231-241. Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizingantibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizingand AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblottingprocedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.
INTRODUCTION
Bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae infects virtually all ruminant species and is arthropod borne (Murphy et al., 1971 ). Bluetongue virus contains 10 segments of double-stranded ribonucleic *Supported in part with funds provided by the U.S.D.A. under the Animal Health Act of 1977, Public Law 95-113, and a U.S.D.A. special grant No. 83-CRSR-2-2186.
0378-1135/88/$03.50
© 1988 Elsevier Science Publishers B.V.
232 acid which code for 7 structural ( P1, P2, P3, P4, P5, P6, P7) and 4 non-structural (NS1, NS2, P8, P8A) polypeptides (Grubman et al., 1983; Sanger and Mertens, 1983). The virus capsid is composed of 4 major structural polypeptides (P2, P3, P5, P7 ) and 3 minor structural polypeptides (P1, P4, P6). Polypeptides P1, P3, P4, P6 and P7 are found in the core particle and P2 and P5 form a diffuse outer coat (Verwoerd et al., 1972; Martin et al., 1973). The antigens responsible for type-specifity and group-specificity are located on polypeptides P2 and P7, respectively (Huismans and Erasmus, 1981 ). The viral protein-specific immune responses of sheep and other ruminant species to BTV infections are poorly characterized. Furthermore, the immune response (s) which constitutes protective immunity and those responsible for clearance of virus from an infected animal are poorly defined. Therefore, studies directed at defining specific immune responses to individual virus proteins and correlating such responses with protective immunity and virus clearance are needed. The immune responses, and their viral protein specificities, responsible for protective immunity and virus clearance are poorly defined. Development of virus-neutralizing antibodies has classically been associated with protective immunity to BTV (Neitz, 1948; Howell, 1960; Luedke and Jochim, 1968a; Campbell, 1985). However, the protective role of such antibody has been questioned owing to: (i) the ability of infectious virus and neutralizing antibody to coexist in an infected animal (Luedke et al., 1969; Luedke, 1969) ; (ii) identification of sheep resistant to BTV infection in the absence of neutralizing antibody (Jochim et al., 1965; Luedke and Jochim, 1968b; Stott et al., 1985); (iii) demonstration of protection via cell-mediated immunity and/or non-neutralizing antibody (Jeggo et al., 1985 ). This report represents a study directed at defining the temporal acquisition of humoral immune response ( s ), and their viral protein specificities, of sheep naturally infected with BTV. In addition to characterization of the viral protein (s) that induces antibody, the potential diagnostic value of immunoblotting is discussed. MATERIALSAND METHODS Animals
Eleven yearling sheep were assembled as part of a multispecies sentinel herd for studying the epidemiology of BTV in an endemic area. Sheep were bled for virus isolation over 6 months; every 2 weeks from April to June, then weekly from June through October. Additional antiserum was obtained, for the purpose of determining assay specificity, as follows: (i) antiserum specific for epizootic haemorrhagic disease virus (EHDV) serotype 1 was produced by infection of a calf with one virus inoculation; (ii) antiserum specific for BTV serotype 17 was produced
233
by infection of sheep with either one inoculation or two inoculations at a 42day interval; (iii) antiserum specific for BTV serotype 13 was produced by hyperimmunization (four inoculations at 30-day intervals) of a rabbit with partially-purified virus in Freund's incomplete adjuvant; (iv) specific pathogen-free (SPF) sheep serum.
Virus isolation Heparinized blood samples were obtained from all sheep for the purpose of virus isolation. Blood cells were washed twice in phosphate-buffered saline ( P B S ) , disrupted by sonication, diluted 1:10 in PBS and 0.1 ml inoculated intravenously into each of six 11-day-old embryonating chicken eggs. Embryos that died between 2 and 6 days post-inoculation (p.i.) were harvested, emacerated, sonicated and applied to cell cultures (VERO) for virus identification.
Serology Agar gel immunodiffusion (AGID) (Pearson and Jochim, 1979) was conducted on all serum samples (antigen supplied by United States Department of Agriculture's National Veterinary Services Laboratory) for identifying BTV group-specific antibodies. BTV serotype-specific antibodies were identified by virus neutralization (SN). Constant virus (100 tissue culture-infectious doses) and varying serum concentrations (serial 10-fold dilutions) were used in the SN test; 80% reduction of cytopathic effect was considered positive for BTV neutralizing antibody.
Immunoblotting BTV antigen for immunoblotting was prepared as follows. Virus was propagated in baby-hamster kidney (BHK-21) cells and partially purified. Virus was extracted from the cell pellet by sonication in 2 mM tris-HC1 containing 0.5% Triton X-100, centrifuged (10 000 × g ) to remove cell debris and pelleted through a 40% (w/v) sucrose cushion (76 000×g). Partially-purified virus was adjusted to a protein concentration of 1 mg m l - 1 and subjected to discontinuous sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions (Laemmli, 1970). Fifty micrograms of virus was applied to each gel using an ll.5-cm sampleapplication well. Electrophoretic transfer of the BTV proteins from the gel to the blotting membrane and the subsequent immunodetection was done as described (Towbin et al., 1979; Akison et al., 1987). Immunoblots were scanned in reflectance mode at 600 nm in a Shimadzu
234 TABLE I Summary of virologic and serologic results obtained before (pre-vector season ), and after ( vector season), virus isolation Animal
RAM 475 479 483 487 489 492 493 494 481 484
Pre-vector season
Virus isolation
Neut a
AGID b
Blot c
< < < < < < < < < < <
-----+ ---+ +
+ + + + + + + + + + +
1 1 1 1 1 1 1 1 1 1 1
+ + + + + + + + + ---
Vector season Neut
AGID
Blot
3 2 3 3 3 3 3 3 3 3
+ + + + + + + + + +
+ + + + + + + + + + (~
3
+
+ ,t
aNeut -- logm of virus-neutralizing antibody titer. bAGID = agar gel immunodiffusion. ¢Blot -- antibody detection by western immunoblotting. dThe relative amount of BTV protein-specific antibody remained unchanged from pre-exposure. All other sheep had demonstrable increases in the relative amount of antibody as determined by immunoblotting (see Table II). T A B L E II Protein-specific antibody response of sentinal sheep to bluetongue virus Animal
RAM 475 479 483 487 489 492 493 494 481 484
BTV serotype isolated
BTV polypeptides 2
3
5
NS1
NS2
6
7
L M W ~'
17 17 11 11 11 17 11 11 11
2/4 ~ 2/2 1/2 2/3 1/3 1/1 2/3 2/2 1/3 1/1 1/1
1/4 2/2 1/1 3/4 1/1 1/1 1/2 1/1 1/1 1/1 1/1
3/4 2/2 1/3 2/4 2/3 1/1 2/3 1/1 2/3 2/2 1/1
1/2 -/-/2 1/4 -/1 1/4 1/2 0/2 2/4 /1/1
-/4 -/4 -/3 -/4 -/4 -/-/4 -//4 //-
-/2 -/-/-/1 -/-/-/1 -/-/2 /-/-
2/3 3/3 2/2 2/4 2/3 2/2 2/4 2/2 2/2 1/1 1/1
/1 -/2 -/3 2/4 -/1 2/3 1/4 1/1 2/3 1/1 /-
~'LMW, low molecular weight BTV protein. bSerological (immunoblotting) results are given before and after virus isolation, e.g., before isolation/after virus isolation. Numeric values represent densitometer scans of blots as follows: - = < 3 mm; 1 -- 3-5 m m peak; 2 = 6-15 m m peak; 3 = 16-30 m m peak; 4 = > 30 m m peak.
235
dual-wavelength TLC scanner, model CS-930, with a Shimadzu data recorder, model DR-2 (Shimadzu Corporation, Kyoto, Japan) set in reflectance mode. RESULTS
The virologic and serologic results are briefly summarized in Table I. Prior to the natural introduction of BTV into the flock, no sheep had detectable BTV neutralizing antibodies, 3 sheep were serologically positive as determined by AGID and all sheep had antibodies specific for a minimum of one BTV protein as determined by western immunoblotting. The BTV protein specificities of these antibodies are given in Table II. Nine of 11 sheep developed viremia following the emergence of the vector (Table I); BTV Serotypes 11 and 17 were identified (Table II). The postviremia serologic results are given in Table I. All sheep developed antibody as A
B
C
DE
jP2 -'P3
p3k p2 F
PS-------NS2 P7,,_
G H i J
F
_,,P2 -'P5
-P5 -NS1 .jNS2 -"P6 -P7
X/'-
m
Fig. 1. Western immunoblot of bluetongue virus polypeptides using BTV Serotype 17 sheep antiserum. Viral proteins identified are given in the margins. X represents a non-characterized viral protein. Lane A; BTV 17 polypeptides detected with amido black protein stain; lane B, BTV 17 polypeptides detected with ram antisera specific for BTV Serotype 17; lane C, BTV 11 polypeptides detected with the same antisera as in Lane B; lane D, BTV 17 polypeptides detected with Sheep 489 antisera specific for BTV Serotype 11; lane E, BTV 11 polypeptides detected with the same antisera as in Lane D; lane F, BTV 11 polypeptides detected with amido black protein stain; lane G, BTV 17 polypeptides probed with specific pathogen free (SAF) negative sheep sera; lane H, BTV 11 polypeptides probed with the same serum as in Lane G.
236 N82 VP3
VP5
'
BTV
17
E VP6 VP7
j,
SCAN
v, l
/VP3
NS2
l
o,v,1
=
Fig. 2. Densitometer tracing of immunoblots conducted by incubation of antisera (specific for BTV 17 ) with blots of BTV 11 and 17. Peaks are labelled with corresponding BTV polypeptides.
A B C D E F G H
................
I J
~P3 ~P2
--NS1 __~NS2 :3-- P6 --P7
i
i
Fig. 3. Western immunoblots of bluetongue virus serotype 17 polypeptides using serum obtained from the ram before, during and after infection with BTV Serotype 17. Lanes A and B, preexposure sera; lanes C, D and G, virus detectable in blood; lanes E, F, H, I and J, no virus detectable in blood; lanes B - H represent 1-week intervals.
237
A B C D E
FG
H
I J
~P5
~P2 --P5 --NSI --NS2
~P7
"--~X
t LMW~ Fig. 4. Western immunoblotsof bluetonguevirus serotype 17 polypeptidesusingserum from sheep 483 obtained before, during and after natural infection with BTV Serotype 11. LMW represents low molecularweight virus proteins that have not been previouslydefined and X represents an additional non-characterizedviral protein. Lanes A and B, pre-exposure sera; lanes C and D, virus detected in blood; lanes E-J, no virus detected in blood; lanes C-I represent 1-weekintervals. determined by AGID (those previously positive remained positive) and virus neutralization, including the 2 sheep t h a t did not develop a detectable viremia. While all 11 sheep continued to express antibodies specific for BTV protein ( s ), only the 9 sheep t h a t developed viremia demonstrated an increase in antibody to individual proteins (Table II). Since western immunoblotting has not previously been described, relative to assaying humoral immune responses of r u m i n a n t species to BTV infection, additional attention was given to this aspect. Assay specificity was determined by blotting multiple serotypes of BTV and E H D V (an orbivirus related to BTV ) followed by immunoassay using ovine antisera specific for BTV, hyperimmune rabbit antisera specific for BTV, bovine antisera specific for E H D V and SPF negative control sheep serum. No serologic cross-reactions were ob-
238 served between BTV and EHDV using the ruminant (bovine or sheep) antisera and no reaction was observed using the SPF negative control sheep sera. Serologic cross-reactions between BTV and EHDV were observed using EHDV proteins and hyperimmune rabbit sera specific for BTV. In the latter case, BTV rabbit antisera recognized up to 4 EHDV proteins. Application of BTV Serotype 17-specific antisera to blots of BTV Serotypes 11 and 17 demonstrated no specificity (Fig. 1 ). The only difference in the immunoblot profile is that VP-2 of serotype 17 migrates ahead of VP-3 during SDS-PAGE. This is well illustrated by comparing the densitometer tracings of immunoblots conducted by incubation of antisera (specific for BTV 17) with blots of BTV 11 and 17 (Fig. 2). Because of the lack of BTV serotype specificity in the western immunoblot, all sheep sera were tested on blots of Serotype 17. Extensive animal variability was observed in development, or relative increase, of viral protein-specific IgG over the course of this study. Figs. 3 and 4 illustrate the serologic profiles of two animals over time. Both the ram and Sheep 483 developed strong antibody responses to the virus-specific Proteins 2, 3, 5, NS2 and 7. While the ram also developed antibodies specific for VP6 (Sheep 483 did not), Sheep 483 developed high titer antibody specific for NS1 and multiple low molecular weight (LMW) viral proteins. This animal variability was a general feature of the immunoblots conducted on all sheep and is illustrated in Table II. DISCUSSION The primary intent of this study was to define the temporal acquisition of viral protein-specific humoral immune responses in sheep, following natural exposure to BTV. This was realized by using a western immunoblotting procedure. The standard serologic techniques, AGID and virus neutralization, were also conducted for comparative purposes. Specificity of the immunoblotting procedure was determined prior to initiation of the study. Ovine antisera specific for BTV and bovine antisera specific for EHDV were applied in 2-way crosses to blots of purified BTV and EHDV proteins. No cross-reactions were observed between these two related orbiviruses. However, 1-way cross-reactions were observed when hyperimmune BTV antiserum was applied to EHDV proteins; the reverse assay was not conducted. Previous studies using AGID and immune precipitations have demonstrated that some BTV and EHDV proteins share antigenic properties (Huismans et al., 1979; Huismans and Els, 1979; Appleton and Letchworth, 1983). These studies employed hyperimmune serum produced in laboratory animals ( mice, guinea pigs and rabbits). The absence of detectable cross-reactions between BTV and EHDV using ruminant sera may be due to species differences (ruminant versus laboratory animal) and/or hyperimmunization. The western immunoblotting assay demonstrated no serotype specificity.
239
Ovine antisera, specific for B T V Serotype 17, reacted with other B T V serotypes in immunoblots in a similar manner (Fig. 1). The ability of serotypespecific antisera to bind equally well to virus protein 2 (VP-2) derived from different serotypes demonstrates a minimum of one antigenic epitope on this protein that is not involved in neutralization. The neutralization epitope on VP-2 was probably destroyed under the denaturing conditions (SDS and 2mercaptoethanol) of the PAGE procedure employed prior to blotting. The lack of correlation between virus-neutralizing antibody and ability of antibody to bind to VP-2 in the immunoblot was also demonstrated using post-virus exposure sera obtained from the two sheep that did not develop detectable viremia. Neither animal demonstrated an increase in antibody capable of binding VP-2 in the immunoblot but an increase in antibody capable of binding the neutralizing epitope in VP-2 was demonstrated by the neutralization test. The 2 assays appear to identify different epitopes on VP-2. The sheep described in this study were assembled as a sentinel flock for the purpose of studying B T V infection and associated serologic responses under naturally occurring field conditions. The sheep were assembled in the spring months, prior to emergence of the B T V vector Culicoides variipennis, and thus prior to the time that virus was transmitted in the environment. The serologic profile of these sheep, before and after B T V exposure, is summarized in Table I; the development of viremia is also illustrated. The superior sensitivity of the western immunoblot, relative to AGID and virus neutralization, is remarkable. All sheep had pre-existing antibody specific for B T V Proteins 2, 3, 5 and 7. The presence of pre-existing antibody specific for viral proteins NS1 and the low molecular weight proteins was variable; none of the sheep had antibody specific for NS2 or VP6. The presence of BTV-specific antibody in sheep prior to the vector season is not surprising. The animals in this study were born the previous spring in an area considered endemic for bluetongue. It is possible that these animals were subsequently exposed to virus and mounted a limited immune response to a viremia that was of low level due to the presence of colostral antibody. Such a controlled infection could explain the absence of detectable virus-neutralizing antibody 6 months later when the animals were assembled for this study. The basis for the presence of pre-existing AGID antibody in 3 sheep that had no virus-neutralizing antibody is not known. These 3 sheep had no demonstrable neutralizing antibody to B T V Serotypes 10, 11, 13 and 17 or to E H D V Serotypes 1 and 2. Prior exposure to unidentified serotypes of BTV, E H D V or other antigenically-related viruses could be responsible for the AGID antibody. Nine of the 11 sheep developed a demonstrable viremia in late summer, which was at a time of peak vector activity. The 2 sheep that did not have a detectable viremia (Sheep 481 and 484) were probably exposed to BTV. This was indicated by the development of antibody capable of neutralizing B T V Serotype 17; these 2 sheep had pre-existing AGID antibody and since the assay is not
240
quantitative an increase in titer could not be detected. However, neither sheep demonstrated an increase in BTV protein-specific antibody as determined by western immunoblotting. An absence of appreciable virus replication, due to pre-existing humoral and/or cellular immunity, may have been responsible for this lack of increased antibody. These results are further evidence that the neutralization assay detects epitopes on VP-2 different from those detected in the immunoblot. This phenomenon is of importance in that the immunoblot appears to be a sensitive indicator of viremia. Application of this technique to paired sera could indicate the presence of a viremia without requiring virus isolation, thus serving as a valuable diagnostic tool. The fact that the immunoblot is not BTV serotype-specific permits the use of a single virus serotype, negating the requirement for multiple assays. In addition, the immunoblot appeared to be group-specific when assaying ruminant sera; sera from BTV-infected sheep did not cross-react with EHDV proteins and sera from EHDVinfected calves did not cross-react with BTV proteins. Testing of additional animals will be required to confirm these observations. If such group-specificity of the immunoblot is further established, the technique will provide a valuable epidemiologic and diagnostic aid. In summary, western immunoblotting appears to be a sensitive assay for detecting humoral immune responses to BTV. Sheep respond to multiple BTV proteins including 5 structural, 2 non-structural and several non-characterized low molecular weight proteins; the heterogeneity of the response is highly dependent upon the individual animal. The role of these multiple-antibody populations in protective immunity and/or virus clearance are at present unknown and deserve additional research. An additional important aspect of this study is the diagnostic potential of the western immunoblotting procedure. Immunoblotting appears to be a superior assay relative to: (i) determining past exposure to BTV; (ii) providing a BTV group-specific serologic technique with limited or absent cross-reactivity to EHDV; (iii) serving as a sensitive indicator of animal viremia using paired sera.
REFERENCES Adkison, M.A., Stott, J.L. and Osburn, B.I., 1987. Identification of bluetongue virus proteinspecific antibody responses in sheep by immunoblotting. Am. J. Vet. Res., 48:1194-1198. Appleton, J.A. and Letchworth, G.J., 1983. Monocional antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants. Virology, 124: 286-299. Campbell, C.H., 1985. Bluetongue: diagnostic/antigenic interpretation. In: T.L. Barber and M.M. Jochim (Editors), Bluetongue and Related Orbiviruses. Proceedings of an International Symposium, January 16-201984, at Monterey, California, U.S.A. Prog. Clin. Biol. Res., 178: 435-443. Grubman, M.J., Appleton, J.A. and Letchworth, G.J., 1983. Identification of BTV type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology, 131: 355-366.
241 Howell, P.G., 1960. A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 28: 357-363. Huismans, H. and Els, H.J., 1979. Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 92: 397-406. Huismans, H. and Erasmus, B.J., 1981. Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. Vet. Res., 48: 51-58. Huismans, H., Bremer, C.W. and Barber, T.L., 1979. The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J. Vet. Res., 46: 95-104. Jeggo, M.H., Wardley, R.C. and Brownlie, J., 1985. Importance of ovine cytotoxic T cells in protection against bluetongue virus infection. In: T.L. Barber and M.M. Jochim (Editors), Bluetongue and related orbiviruses. Proceedings of an International Symposium, January 16-20 1984, at Monterey, California, U.S.A. Prog. Clin. Biol. Res., 178: 477-487. Jochim, M.M., Leudke, A.J. and Bowne, J.G., 1965. The clinical and immunogenic response of sheep to oral and intradermal administrations of bluetongue virus. Am. J. Vet. Res., 26: 1254-1260. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Luedke, A.J., 1969. Bluetongue in sheep: Viral assay and viremia. Am. J. Vet. Res., 30: 499-509. Luedke, A.J. and Jochim, M.M., 1968a. Clinical and serologic responses in vaccinated sheep given challenge inoculation with isolates of bluetongue virus. Am. J. Vet. Res., 29: 841-851. Luedke, A.J. and Jochim, M.M., 1968b. Bluetongue virus in sheep. Intensification of the clinical response by previous oral administration. Cornell Vet., 58: 48-58. Ludke, A.J., Jochim, M.M. and Jones, R.H., 1969. Bluetongue in cattle: Viremia. Am. J. Vet. Res., 30: 511-516. Martin, S.A., Pett, D.M. and Zweerink, H.J., 1973. Studies on the topography of reovirus and bluetongue virus capsid proteins. J. Virol., 12: 194-198. Murphy, F.A., Borden, E.C., Shope, R.E. and Harrison, A., 1971. Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus. A new taxonomic group. Electron microscopic studies. J. Gen. Virol., 13: 273-288. Neitz, W.O., 1948. Immunological studies on bluetongue in sheep. Onderstepoort J. Vet. Sci. Anim. Ind., 23: 93-136. Pearson, J.E. and Jochim, M.M., 1979. Protocol for the Immunodiffusion Test for Bluetongue. Proc. Annu. Meet. Am. Assoc. Vet. Lab. Diagn., 22: 463-471. Sanger, D.V. and Mertens, P.P.C., 1983. Comparison of type 1 bluetongue virus protein synthesis in vivo and in vitro. In: R.W. Compans and D.H.L. Bishop (Editors), Double-Stranded RNA Viruses. Proceedings of a symposium, October 5-10 1982, at St. Thomas, U.S. Virgin Islands. Elsevier Science Publishing Company, New York, pp. 183-191. Stott, J.L., Barber, T.L. and Osburn, B.I., 1985. Immunologic response of sheep to inactivated and virulent bluetongue. Am. J. Vet. Res., 46: 1043-1049. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Verwoerd, D.W., Els, H.J., DeVilliers, E. and Huismans, H., 1972. Structure of the bluetongue virus capsid. J. Virol., 10: 783-794.
231
Temporal Development of Bluetongue Virus Protein-specific Antibody in Sheep following Natural Infection* M.A. ADKISON 1, J.L. STOTT' and B.I. OSBURN 2
'Department of Microbiology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) 2Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted for publication 6 October 1987)
ABSTRACT Adkison, M.A., Stott, J.L. and Osburn, B.I., 1988. Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection. Vet. Microbiol., 16: 231-241. Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizingantibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizingand AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblottingprocedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.
INTRODUCTION
Bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae infects virtually all ruminant species and is arthropod borne (Murphy et al., 1971 ). Bluetongue virus contains 10 segments of double-stranded ribonucleic *Supported in part with funds provided by the U.S.D.A. under the Animal Health Act of 1977, Public Law 95-113, and a U.S.D.A. special grant No. 83-CRSR-2-2186.
0378-1135/88/$03.50
© 1988 Elsevier Science Publishers B.V.
232 acid which code for 7 structural ( P1, P2, P3, P4, P5, P6, P7) and 4 non-structural (NS1, NS2, P8, P8A) polypeptides (Grubman et al., 1983; Sanger and Mertens, 1983). The virus capsid is composed of 4 major structural polypeptides (P2, P3, P5, P7 ) and 3 minor structural polypeptides (P1, P4, P6). Polypeptides P1, P3, P4, P6 and P7 are found in the core particle and P2 and P5 form a diffuse outer coat (Verwoerd et al., 1972; Martin et al., 1973). The antigens responsible for type-specifity and group-specificity are located on polypeptides P2 and P7, respectively (Huismans and Erasmus, 1981 ). The viral protein-specific immune responses of sheep and other ruminant species to BTV infections are poorly characterized. Furthermore, the immune response (s) which constitutes protective immunity and those responsible for clearance of virus from an infected animal are poorly defined. Therefore, studies directed at defining specific immune responses to individual virus proteins and correlating such responses with protective immunity and virus clearance are needed. The immune responses, and their viral protein specificities, responsible for protective immunity and virus clearance are poorly defined. Development of virus-neutralizing antibodies has classically been associated with protective immunity to BTV (Neitz, 1948; Howell, 1960; Luedke and Jochim, 1968a; Campbell, 1985). However, the protective role of such antibody has been questioned owing to: (i) the ability of infectious virus and neutralizing antibody to coexist in an infected animal (Luedke et al., 1969; Luedke, 1969) ; (ii) identification of sheep resistant to BTV infection in the absence of neutralizing antibody (Jochim et al., 1965; Luedke and Jochim, 1968b; Stott et al., 1985); (iii) demonstration of protection via cell-mediated immunity and/or non-neutralizing antibody (Jeggo et al., 1985 ). This report represents a study directed at defining the temporal acquisition of humoral immune response ( s ), and their viral protein specificities, of sheep naturally infected with BTV. In addition to characterization of the viral protein (s) that induces antibody, the potential diagnostic value of immunoblotting is discussed. MATERIALSAND METHODS Animals
Eleven yearling sheep were assembled as part of a multispecies sentinel herd for studying the epidemiology of BTV in an endemic area. Sheep were bled for virus isolation over 6 months; every 2 weeks from April to June, then weekly from June through October. Additional antiserum was obtained, for the purpose of determining assay specificity, as follows: (i) antiserum specific for epizootic haemorrhagic disease virus (EHDV) serotype 1 was produced by infection of a calf with one virus inoculation; (ii) antiserum specific for BTV serotype 17 was produced
233
by infection of sheep with either one inoculation or two inoculations at a 42day interval; (iii) antiserum specific for BTV serotype 13 was produced by hyperimmunization (four inoculations at 30-day intervals) of a rabbit with partially-purified virus in Freund's incomplete adjuvant; (iv) specific pathogen-free (SPF) sheep serum.
Virus isolation Heparinized blood samples were obtained from all sheep for the purpose of virus isolation. Blood cells were washed twice in phosphate-buffered saline ( P B S ) , disrupted by sonication, diluted 1:10 in PBS and 0.1 ml inoculated intravenously into each of six 11-day-old embryonating chicken eggs. Embryos that died between 2 and 6 days post-inoculation (p.i.) were harvested, emacerated, sonicated and applied to cell cultures (VERO) for virus identification.
Serology Agar gel immunodiffusion (AGID) (Pearson and Jochim, 1979) was conducted on all serum samples (antigen supplied by United States Department of Agriculture's National Veterinary Services Laboratory) for identifying BTV group-specific antibodies. BTV serotype-specific antibodies were identified by virus neutralization (SN). Constant virus (100 tissue culture-infectious doses) and varying serum concentrations (serial 10-fold dilutions) were used in the SN test; 80% reduction of cytopathic effect was considered positive for BTV neutralizing antibody.
Immunoblotting BTV antigen for immunoblotting was prepared as follows. Virus was propagated in baby-hamster kidney (BHK-21) cells and partially purified. Virus was extracted from the cell pellet by sonication in 2 mM tris-HC1 containing 0.5% Triton X-100, centrifuged (10 000 × g ) to remove cell debris and pelleted through a 40% (w/v) sucrose cushion (76 000×g). Partially-purified virus was adjusted to a protein concentration of 1 mg m l - 1 and subjected to discontinuous sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions (Laemmli, 1970). Fifty micrograms of virus was applied to each gel using an ll.5-cm sampleapplication well. Electrophoretic transfer of the BTV proteins from the gel to the blotting membrane and the subsequent immunodetection was done as described (Towbin et al., 1979; Akison et al., 1987). Immunoblots were scanned in reflectance mode at 600 nm in a Shimadzu
234 TABLE I Summary of virologic and serologic results obtained before (pre-vector season ), and after ( vector season), virus isolation Animal
RAM 475 479 483 487 489 492 493 494 481 484
Pre-vector season
Virus isolation
Neut a
AGID b
Blot c
< < < < < < < < < < <
-----+ ---+ +
+ + + + + + + + + + +
1 1 1 1 1 1 1 1 1 1 1
+ + + + + + + + + ---
Vector season Neut
AGID
Blot
3 2 3 3 3 3 3 3 3 3
+ + + + + + + + + +
+ + + + + + + + + + (~
3
+
+ ,t
aNeut -- logm of virus-neutralizing antibody titer. bAGID = agar gel immunodiffusion. ¢Blot -- antibody detection by western immunoblotting. dThe relative amount of BTV protein-specific antibody remained unchanged from pre-exposure. All other sheep had demonstrable increases in the relative amount of antibody as determined by immunoblotting (see Table II). T A B L E II Protein-specific antibody response of sentinal sheep to bluetongue virus Animal
RAM 475 479 483 487 489 492 493 494 481 484
BTV serotype isolated
BTV polypeptides 2
3
5
NS1
NS2
6
7
L M W ~'
17 17 11 11 11 17 11 11 11
2/4 ~ 2/2 1/2 2/3 1/3 1/1 2/3 2/2 1/3 1/1 1/1
1/4 2/2 1/1 3/4 1/1 1/1 1/2 1/1 1/1 1/1 1/1
3/4 2/2 1/3 2/4 2/3 1/1 2/3 1/1 2/3 2/2 1/1
1/2 -/-/2 1/4 -/1 1/4 1/2 0/2 2/4 /1/1
-/4 -/4 -/3 -/4 -/4 -/-/4 -//4 //-
-/2 -/-/-/1 -/-/-/1 -/-/2 /-/-
2/3 3/3 2/2 2/4 2/3 2/2 2/4 2/2 2/2 1/1 1/1
/1 -/2 -/3 2/4 -/1 2/3 1/4 1/1 2/3 1/1 /-
~'LMW, low molecular weight BTV protein. bSerological (immunoblotting) results are given before and after virus isolation, e.g., before isolation/after virus isolation. Numeric values represent densitometer scans of blots as follows: - = < 3 mm; 1 -- 3-5 m m peak; 2 = 6-15 m m peak; 3 = 16-30 m m peak; 4 = > 30 m m peak.
235
dual-wavelength TLC scanner, model CS-930, with a Shimadzu data recorder, model DR-2 (Shimadzu Corporation, Kyoto, Japan) set in reflectance mode. RESULTS
The virologic and serologic results are briefly summarized in Table I. Prior to the natural introduction of BTV into the flock, no sheep had detectable BTV neutralizing antibodies, 3 sheep were serologically positive as determined by AGID and all sheep had antibodies specific for a minimum of one BTV protein as determined by western immunoblotting. The BTV protein specificities of these antibodies are given in Table II. Nine of 11 sheep developed viremia following the emergence of the vector (Table I); BTV Serotypes 11 and 17 were identified (Table II). The postviremia serologic results are given in Table I. All sheep developed antibody as A
B
C
DE
jP2 -'P3
p3k p2 F
PS-------NS2 P7,,_
G H i J
F
_,,P2 -'P5
-P5 -NS1 .jNS2 -"P6 -P7
X/'-
m
Fig. 1. Western immunoblot of bluetongue virus polypeptides using BTV Serotype 17 sheep antiserum. Viral proteins identified are given in the margins. X represents a non-characterized viral protein. Lane A; BTV 17 polypeptides detected with amido black protein stain; lane B, BTV 17 polypeptides detected with ram antisera specific for BTV Serotype 17; lane C, BTV 11 polypeptides detected with the same antisera as in Lane B; lane D, BTV 17 polypeptides detected with Sheep 489 antisera specific for BTV Serotype 11; lane E, BTV 11 polypeptides detected with the same antisera as in Lane D; lane F, BTV 11 polypeptides detected with amido black protein stain; lane G, BTV 17 polypeptides probed with specific pathogen free (SAF) negative sheep sera; lane H, BTV 11 polypeptides probed with the same serum as in Lane G.
236 N82 VP3
VP5
'
BTV
17
E VP6 VP7
j,
SCAN
v, l
/VP3
NS2
l
o,v,1
=
Fig. 2. Densitometer tracing of immunoblots conducted by incubation of antisera (specific for BTV 17 ) with blots of BTV 11 and 17. Peaks are labelled with corresponding BTV polypeptides.
A B C D E F G H
................
I J
~P3 ~P2
--NS1 __~NS2 :3-- P6 --P7
i
i
Fig. 3. Western immunoblots of bluetongue virus serotype 17 polypeptides using serum obtained from the ram before, during and after infection with BTV Serotype 17. Lanes A and B, preexposure sera; lanes C, D and G, virus detectable in blood; lanes E, F, H, I and J, no virus detectable in blood; lanes B - H represent 1-week intervals.
237
A B C D E
FG
H
I J
~P5
~P2 --P5 --NSI --NS2
~P7
"--~X
t LMW~ Fig. 4. Western immunoblotsof bluetonguevirus serotype 17 polypeptidesusingserum from sheep 483 obtained before, during and after natural infection with BTV Serotype 11. LMW represents low molecularweight virus proteins that have not been previouslydefined and X represents an additional non-characterizedviral protein. Lanes A and B, pre-exposure sera; lanes C and D, virus detected in blood; lanes E-J, no virus detected in blood; lanes C-I represent 1-weekintervals. determined by AGID (those previously positive remained positive) and virus neutralization, including the 2 sheep t h a t did not develop a detectable viremia. While all 11 sheep continued to express antibodies specific for BTV protein ( s ), only the 9 sheep t h a t developed viremia demonstrated an increase in antibody to individual proteins (Table II). Since western immunoblotting has not previously been described, relative to assaying humoral immune responses of r u m i n a n t species to BTV infection, additional attention was given to this aspect. Assay specificity was determined by blotting multiple serotypes of BTV and E H D V (an orbivirus related to BTV ) followed by immunoassay using ovine antisera specific for BTV, hyperimmune rabbit antisera specific for BTV, bovine antisera specific for E H D V and SPF negative control sheep serum. No serologic cross-reactions were ob-
238 served between BTV and EHDV using the ruminant (bovine or sheep) antisera and no reaction was observed using the SPF negative control sheep sera. Serologic cross-reactions between BTV and EHDV were observed using EHDV proteins and hyperimmune rabbit sera specific for BTV. In the latter case, BTV rabbit antisera recognized up to 4 EHDV proteins. Application of BTV Serotype 17-specific antisera to blots of BTV Serotypes 11 and 17 demonstrated no specificity (Fig. 1 ). The only difference in the immunoblot profile is that VP-2 of serotype 17 migrates ahead of VP-3 during SDS-PAGE. This is well illustrated by comparing the densitometer tracings of immunoblots conducted by incubation of antisera (specific for BTV 17) with blots of BTV 11 and 17 (Fig. 2). Because of the lack of BTV serotype specificity in the western immunoblot, all sheep sera were tested on blots of Serotype 17. Extensive animal variability was observed in development, or relative increase, of viral protein-specific IgG over the course of this study. Figs. 3 and 4 illustrate the serologic profiles of two animals over time. Both the ram and Sheep 483 developed strong antibody responses to the virus-specific Proteins 2, 3, 5, NS2 and 7. While the ram also developed antibodies specific for VP6 (Sheep 483 did not), Sheep 483 developed high titer antibody specific for NS1 and multiple low molecular weight (LMW) viral proteins. This animal variability was a general feature of the immunoblots conducted on all sheep and is illustrated in Table II. DISCUSSION The primary intent of this study was to define the temporal acquisition of viral protein-specific humoral immune responses in sheep, following natural exposure to BTV. This was realized by using a western immunoblotting procedure. The standard serologic techniques, AGID and virus neutralization, were also conducted for comparative purposes. Specificity of the immunoblotting procedure was determined prior to initiation of the study. Ovine antisera specific for BTV and bovine antisera specific for EHDV were applied in 2-way crosses to blots of purified BTV and EHDV proteins. No cross-reactions were observed between these two related orbiviruses. However, 1-way cross-reactions were observed when hyperimmune BTV antiserum was applied to EHDV proteins; the reverse assay was not conducted. Previous studies using AGID and immune precipitations have demonstrated that some BTV and EHDV proteins share antigenic properties (Huismans et al., 1979; Huismans and Els, 1979; Appleton and Letchworth, 1983). These studies employed hyperimmune serum produced in laboratory animals ( mice, guinea pigs and rabbits). The absence of detectable cross-reactions between BTV and EHDV using ruminant sera may be due to species differences (ruminant versus laboratory animal) and/or hyperimmunization. The western immunoblotting assay demonstrated no serotype specificity.
239
Ovine antisera, specific for B T V Serotype 17, reacted with other B T V serotypes in immunoblots in a similar manner (Fig. 1). The ability of serotypespecific antisera to bind equally well to virus protein 2 (VP-2) derived from different serotypes demonstrates a minimum of one antigenic epitope on this protein that is not involved in neutralization. The neutralization epitope on VP-2 was probably destroyed under the denaturing conditions (SDS and 2mercaptoethanol) of the PAGE procedure employed prior to blotting. The lack of correlation between virus-neutralizing antibody and ability of antibody to bind to VP-2 in the immunoblot was also demonstrated using post-virus exposure sera obtained from the two sheep that did not develop detectable viremia. Neither animal demonstrated an increase in antibody capable of binding VP-2 in the immunoblot but an increase in antibody capable of binding the neutralizing epitope in VP-2 was demonstrated by the neutralization test. The 2 assays appear to identify different epitopes on VP-2. The sheep described in this study were assembled as a sentinel flock for the purpose of studying B T V infection and associated serologic responses under naturally occurring field conditions. The sheep were assembled in the spring months, prior to emergence of the B T V vector Culicoides variipennis, and thus prior to the time that virus was transmitted in the environment. The serologic profile of these sheep, before and after B T V exposure, is summarized in Table I; the development of viremia is also illustrated. The superior sensitivity of the western immunoblot, relative to AGID and virus neutralization, is remarkable. All sheep had pre-existing antibody specific for B T V Proteins 2, 3, 5 and 7. The presence of pre-existing antibody specific for viral proteins NS1 and the low molecular weight proteins was variable; none of the sheep had antibody specific for NS2 or VP6. The presence of BTV-specific antibody in sheep prior to the vector season is not surprising. The animals in this study were born the previous spring in an area considered endemic for bluetongue. It is possible that these animals were subsequently exposed to virus and mounted a limited immune response to a viremia that was of low level due to the presence of colostral antibody. Such a controlled infection could explain the absence of detectable virus-neutralizing antibody 6 months later when the animals were assembled for this study. The basis for the presence of pre-existing AGID antibody in 3 sheep that had no virus-neutralizing antibody is not known. These 3 sheep had no demonstrable neutralizing antibody to B T V Serotypes 10, 11, 13 and 17 or to E H D V Serotypes 1 and 2. Prior exposure to unidentified serotypes of BTV, E H D V or other antigenically-related viruses could be responsible for the AGID antibody. Nine of the 11 sheep developed a demonstrable viremia in late summer, which was at a time of peak vector activity. The 2 sheep that did not have a detectable viremia (Sheep 481 and 484) were probably exposed to BTV. This was indicated by the development of antibody capable of neutralizing B T V Serotype 17; these 2 sheep had pre-existing AGID antibody and since the assay is not
240
quantitative an increase in titer could not be detected. However, neither sheep demonstrated an increase in BTV protein-specific antibody as determined by western immunoblotting. An absence of appreciable virus replication, due to pre-existing humoral and/or cellular immunity, may have been responsible for this lack of increased antibody. These results are further evidence that the neutralization assay detects epitopes on VP-2 different from those detected in the immunoblot. This phenomenon is of importance in that the immunoblot appears to be a sensitive indicator of viremia. Application of this technique to paired sera could indicate the presence of a viremia without requiring virus isolation, thus serving as a valuable diagnostic tool. The fact that the immunoblot is not BTV serotype-specific permits the use of a single virus serotype, negating the requirement for multiple assays. In addition, the immunoblot appeared to be group-specific when assaying ruminant sera; sera from BTV-infected sheep did not cross-react with EHDV proteins and sera from EHDVinfected calves did not cross-react with BTV proteins. Testing of additional animals will be required to confirm these observations. If such group-specificity of the immunoblot is further established, the technique will provide a valuable epidemiologic and diagnostic aid. In summary, western immunoblotting appears to be a sensitive assay for detecting humoral immune responses to BTV. Sheep respond to multiple BTV proteins including 5 structural, 2 non-structural and several non-characterized low molecular weight proteins; the heterogeneity of the response is highly dependent upon the individual animal. The role of these multiple-antibody populations in protective immunity and/or virus clearance are at present unknown and deserve additional research. An additional important aspect of this study is the diagnostic potential of the western immunoblotting procedure. Immunoblotting appears to be a superior assay relative to: (i) determining past exposure to BTV; (ii) providing a BTV group-specific serologic technique with limited or absent cross-reactivity to EHDV; (iii) serving as a sensitive indicator of animal viremia using paired sera.
REFERENCES Adkison, M.A., Stott, J.L. and Osburn, B.I., 1987. Identification of bluetongue virus proteinspecific antibody responses in sheep by immunoblotting. Am. J. Vet. Res., 48:1194-1198. Appleton, J.A. and Letchworth, G.J., 1983. Monocional antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants. Virology, 124: 286-299. Campbell, C.H., 1985. Bluetongue: diagnostic/antigenic interpretation. In: T.L. Barber and M.M. Jochim (Editors), Bluetongue and Related Orbiviruses. Proceedings of an International Symposium, January 16-201984, at Monterey, California, U.S.A. Prog. Clin. Biol. Res., 178: 435-443. Grubman, M.J., Appleton, J.A. and Letchworth, G.J., 1983. Identification of BTV type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology, 131: 355-366.
241 Howell, P.G., 1960. A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 28: 357-363. Huismans, H. and Els, H.J., 1979. Characterization of the tubules associated with the replication of three different orbiviruses. Virology, 92: 397-406. Huismans, H. and Erasmus, B.J., 1981. Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. Vet. Res., 48: 51-58. Huismans, H., Bremer, C.W. and Barber, T.L., 1979. The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoort J. Vet. Res., 46: 95-104. Jeggo, M.H., Wardley, R.C. and Brownlie, J., 1985. Importance of ovine cytotoxic T cells in protection against bluetongue virus infection. In: T.L. Barber and M.M. Jochim (Editors), Bluetongue and related orbiviruses. Proceedings of an International Symposium, January 16-20 1984, at Monterey, California, U.S.A. Prog. Clin. Biol. Res., 178: 477-487. Jochim, M.M., Leudke, A.J. and Bowne, J.G., 1965. The clinical and immunogenic response of sheep to oral and intradermal administrations of bluetongue virus. Am. J. Vet. Res., 26: 1254-1260. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Luedke, A.J., 1969. Bluetongue in sheep: Viral assay and viremia. Am. J. Vet. Res., 30: 499-509. Luedke, A.J. and Jochim, M.M., 1968a. Clinical and serologic responses in vaccinated sheep given challenge inoculation with isolates of bluetongue virus. Am. J. Vet. Res., 29: 841-851. Luedke, A.J. and Jochim, M.M., 1968b. Bluetongue virus in sheep. Intensification of the clinical response by previous oral administration. Cornell Vet., 58: 48-58. Ludke, A.J., Jochim, M.M. and Jones, R.H., 1969. Bluetongue in cattle: Viremia. Am. J. Vet. Res., 30: 511-516. Martin, S.A., Pett, D.M. and Zweerink, H.J., 1973. Studies on the topography of reovirus and bluetongue virus capsid proteins. J. Virol., 12: 194-198. Murphy, F.A., Borden, E.C., Shope, R.E. and Harrison, A., 1971. Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus. A new taxonomic group. Electron microscopic studies. J. Gen. Virol., 13: 273-288. Neitz, W.O., 1948. Immunological studies on bluetongue in sheep. Onderstepoort J. Vet. Sci. Anim. Ind., 23: 93-136. Pearson, J.E. and Jochim, M.M., 1979. Protocol for the Immunodiffusion Test for Bluetongue. Proc. Annu. Meet. Am. Assoc. Vet. Lab. Diagn., 22: 463-471. Sanger, D.V. and Mertens, P.P.C., 1983. Comparison of type 1 bluetongue virus protein synthesis in vivo and in vitro. In: R.W. Compans and D.H.L. Bishop (Editors), Double-Stranded RNA Viruses. Proceedings of a symposium, October 5-10 1982, at St. Thomas, U.S. Virgin Islands. Elsevier Science Publishing Company, New York, pp. 183-191. Stott, J.L., Barber, T.L. and Osburn, B.I., 1985. Immunologic response of sheep to inactivated and virulent bluetongue. Am. J. Vet. Res., 46: 1043-1049. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Verwoerd, D.W., Els, H.J., DeVilliers, E. and Huismans, H., 1972. Structure of the bluetongue virus capsid. J. Virol., 10: 783-794.