Veterinary Microbiology, 33 ( 1992 ) 143-153 Elsevier Science Publishers B.V., Amsterdam
143
Diagnostic methods for african horsesickness virus using monoclonal antibodies to structural and non-structural proteins Ana I. Ranz, Julita G. Miguet, Carmen Anaya, Angel Venteo, Elena Cort6s, Carmen Vela and Antonio Sanz Immunologia Y Genetica Aplicada S.A. (lngenasa) Madrid. Spain (Accepted 26 June 1992)
ABSTRACT Ranz, A.I., Miguet, J.G., Anaya, C., Venteo, A., Cort6s, E., Vela, C. and Sanz, A., 1992. Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins. Vet. Microbiol., 33: 143-153. A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the nonstructural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 ~1. The NS1specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.
INTRODUCTION
African horse sickness (AHS) is an arthropod-borne disease of Equidae, caused by a dsRNA orbivirus (AHSV) of the Reoviridae Family (Verwoerd et al, 1979). The infection of horses is characterized by a high mortality. The disease is endemic in central Africa, although outbreaks have occurred in North Africa and Southern Europe (Spain and Portugal) in recent years (1987-1991). Only one serotype, AHSV-4, has been isolated in Spain and Portugal during these outbreaks. The virus has a genome consisting of 10 segments of double-stranded RNA (Oellerman et al, 1970), each of which encodes at least one polypeptide. There are seven structural proteins, which form Correspondence to: A. Sanz, Immunologia y Genetica Aplicada S.A. (Ingenase-Hnos. Garcia Noblejas, 4 l-2°-28037-Madrid, Spain.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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a double-shelled virus particle. The outer capsid is composed ofthe two major proteins (VP2 and VP5), which are responsible for the viral neutralization and antigenic variability, whereas the inner capsid is composed of two major (VP3 and VP7), and three minor (VPI, VP4 and VP6) proteins. VP3 and VP7 are highly conserved among the nine AHSV serotypes (Oellerman et al., 1970; Bremer, 1976). AHSV also induces three non-structural proteins (NS 1, NS2 and NS3). A confirmed diagnosis of AHSV infection requires isolation and identification of the virus. This has usually been achieved by passing the putative virus in mouse brains (Howell, 1962) or cell cultures (Mirchamsy and Taslimi, 1963; Ozawa and Hazrati, 1964), and serological typing done by plaque reduction neutralization (Hopkins et al., 1966). However these methods are time consuming and require viable virus. The economic implications of outbreaks of AHSV make it essential to have rapid, sensitive and specific diagnostic tools. The availability of ASHV-specific monoclonal antibodies (MAbs) will be useful to develop diagnostic methods. In this paper we present the characterization of MAbs specific for AHSV-4 proteins, VP2, VP7 and NSI. A double antibody sandwich ELISA (ELISA-DAS) has been developed using VP7-specific MAbs, for the detection of AHSV in spleens from infected animals, as well as an immunofluorescence assay on AHSV-infected Vero cells using NSl-specific MAbs. In addition, these MAbs have been useful to study the antigenic structure of the outer capsid protein VP2. MATERIALS AND METHODS
Cells and virus Vero cells (ATCC) were grown in Dulbecco modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum ( FCS ). Hybridomas were grown in RPMI medium containing 15% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.4/IM aminopterine, 16 mM thymidine and antibiotics (penicillin and streptomycin). Cells were maintained in 8% CO2 atmosphere, The AHS virus serotype 4, used in this study, was isolated from the spleen of an infected horse and cloned twice by plaque isolation in Vero cells. Titres were determined by plaque assay on Vero monolayers as previously described (Oellermann, 1970). Virus was propagated in 850 cm 2 roller bottles of Vero cell cultures and purified as described for bluetongue virus by Mertens et al (1987). Production of hybridoma cell lines Hybridoma lines were obtained by fusion of spleen cells from ASHV immunized mice with the non-secreting myeloma cell line P3X63 Ag 8.653 (Kearny et al, 1979) following the procedure described by Nowinski et al.
DIAGNOSTIC METHODS FOR AFRICAN HORSESICKNESS VIRUS
145
( 1979 ) with slight modifications (Sanz et al. 1985 ). Screening of the hybrids was carried out by testing supernatants in an indirect ELISA using purified virus as antigen. MAbs were purified from ascitic fluid by HPLC (System Gold, Beckman ).
Immunoprecipitation and binding analysis The specificity of the MAbs was determined by two procedures, immunoprecipitation and binding of antigen to MAb-coated plates (immunoadsorption) as previously described (Melero and Gonz~ilez-Rodriguez, 1984; Sanz et al, 1985).
lm m unofluorescence studies Vero cells grown in micorslide culture chambers (Miles Scientific) were infected with ASH virus at multiplicity of infection (moi) 5. At different hours after infection, the cells were washed, fixed with methanol-acetone ( 1:1 ) and reacted with hybridoma supernatants containing the indicated MAb. After washing, cells were covered with fluoresceinated goat anti-mouse immunoglobulins (Sigma). Immunofluorescence was viewed with a Nikon Diaphot microscope equipped with epifluorescence
Immunoblotting analysis of MAbs AHS virus proteins from purified virions were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (Towbin et al, 1979). Membranes were saturated with 3% (w/v) non-fat dry milk in Tris buffered saline for 1 h, at room temperature. The reactivity of MAbs with polypeptides was detected with a biotin conjugated goat anti-mouse IGg (H + L) antibody (Sigma) and alkaline phosphatase-labelled streptavidin (Boehringer), using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro-blue tetrazolium ) as substrates.
Competitive binding ELISA Biotin (biotinamidocaproate N-hydroxysuccinande ester, Sigma) was conjugated as described (Bayer and Wilchek, 1980). MAbs were used in reciprocal competitive ELISA to eliminate the effects of antibody avidity on the results. 96-well plates were coated with 1/tg/well of purified virus, and then washed with phosphate buffered saline (PBS) containing 0.05% Tween 20. Competitions were performed with 10 fold dilutions of the purified MAb. The MAb was added to antigen-coated wells and allowed to react for 1 h at 37°C in the presence of a biotinylated MAb added at a predetermined optimal concentration. After adding streptavidin horseradish peroxidase-labelled (30 min, RT) and chromogen/substrate solution containing 40 mM 3-dimethyl-aminobenzoic acid (DMAB, Sigma), 0.78 mM 3-methyl-2-benzothiazolinone (MBTH, Sigma) and 0.003% H202 in phosphate buffer pH 7,
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the absorbance of each well was measured in a micro ELISA autoreader (Flow) at 600 nm. Alternatively, o-phenylendiamine/H202 was used as chromogen/substrate and the absorbance measured at 450 nm.
Neutralization test Mabs alone or mixtures of MAbs were used in the neutralization test. Neutralization was determined by 90% plaque forming units (pfu) inhibition test. A virus dilution containing 100 pfu was mixed with various dilutions of MAbs (in presence or absence of rabbit anti-mouse immunoglobulins antibody, Sigma) and incubated overnight at 4°C plus 4 h at 37°C to increase the sentivity of the test. Virus adsorption to Vero cells in 24-well plates (Costar) was allowed to proceed at 37 °C for 2 h. The inoculum was removed and cells were covered with the agar overlay m e d i u m (DMEM, 1% agarose and 2% FCS). Five days after plates were fixed and stained with an aqueous alcoholic solution of crystal violet.
Double antibody sandwich Enzyme-Linked immunosorbent assay (ELISADAS) ELISA-DAS was used to test the ability of different MAbs to detect AHS virus in spleen extracts from infected horses. Briefly, wells were coated at various concentrations with different MAbs specific for AHS virus. One hundred/A of spleen extract, prepared by homogenizing 1 g of spleen in 10 ml of PBS, was added to the wells previously coated with antibody and incubated for 60 min at 37°C. U n b o u n d antigen was washed out. Bound antigen was detected by adding MAb conjugated to biotin and incubating 1 h at 37°C. The assay was developed by incubating with streptavidin-horseradish peroxidase and chromogen/substrate as described above. Reactions were considered positive when the OD values were three or more times greater than the mean of the OD obtained with negative samples. Absorbance values between 2 and 3 times greater than the mean of the negative samples were considered doubtful. RESULTS
Preparation and characterization of MAbs Thirty two hybridomas were derived from four fusion experiments. After cloning and stabilization MAbs were produced in ascitic fluid and further characterized by immunoprecipitation, immunoblotting, immunofluorescence staining of AHS virus-infected Vero cells, and virus neutralization. The reactivity of MAbs with purified AHSV virions and AHSV-infected cell lysate was determined by western blotting and immunoprecipitation respectively. Twenty MAbs reacted with VP2 protein, four MAbs recognized VP7 and eight MAbs reacted with NS 1. Figure 1 shows the proteins selected
DIAGNOSTICMETHODSFOR AFRICANHORSESICKNESSVIRUS
1
2
3
4
5
6
7
8
9
147
10 11
12
200 q 100 92.5
6,
~ ~
~ ~ '
'~
VP2
9 41D 6 NSl
4e
B ~
30
~
~
VP7
I
Fig. 1. Immunoprecipitation assay of 35S-methionine labelled AHSV proteins by MAbs. Line I, 14C-molecular weight markers. Lines 2 to 4 MAbs to VP2 (6DG2, 8AF8, 8DA6). Lines 5 to 7, MAbs to NS1 (5BB12, 5AF6, 6CA3). Lines 8 to 10 MAbs to VP7 (4AH9, 8DF4, 8DB6). Line 1 I MAb to rotavirus VP6 and line 12 mouse antiserum to AHSV.
by some MAbs from AHSV-infected cell lysate in immunoprecipitation assay. Polyclonal antibodies reacted with multiple AHS virus polypetides (lane 12 ). No reactivity was observed between MAbs with uninfected Vero cell lysate (data not shown). VP7-specific MAbs did not react in immunoblotting with the denatured protein. On the other hand eight VP2-specific reacted with denatured protein (data not shown). The removal of reducer agent did not restore the activity of non-reactive MAbs.
Reactivity of MAbs with intracellular antigens specified by AHS virus The reactivity of MAbs with AHSV-infected Vero cells was detected by immunofluorescence and is shown in Fig. 2. Three different fluorescence pat-
148
A.I. RANZ ET AL.
O
0
Fig. 2. lmmunofluorescence patterns of selected MAbs reacting with AHS virus-infected (A, B, C) and uninfected Vero cells (A', B', C'). A, A' Mab 8AF8 (VP2); B, B' Mab 8DB6 (VPT); C,C', Mab 5CGI0 ( N S I ) .
D I A G N O S T I C METHODS FOR AFRICAN HORSESICKNESS VIRUS
149
terns were observed: (i) diffuse cytoplasmic and perinuclear fluorescence; this pattern was obtained with the VP2-specific MAbs (Fig. 2A); (ii) fluorescence covering perinuclear and discrete cytoplasmic areas; this pattern was characteristic of the VP7-specific MAb (Fig. 2B); and (iii) staining of tubule-like structures; this pattern was characteristic of NS 1-specific MAbs (Fig. 2C) and was observed earlier than the others (at 6 hours after infection).
Detection of AHSV antigen in biological samples by ELISA-DAS Firstly, we investigated which combination of MAbs would be suited for a ELISA-DAS for the detection of antigen. Wells were coated with 1/tg of purified VP7-specific MAbs and, after incubating with purified antigen, combinations of biotin-labelled VP7-specific MAbs were added to each well. The highest sensitivity was observed when MAbs 4AH9 and 8DF4 were used as the capture antibodies and MAb 8DB6 as the labelled antibody. The sensitivity of the assay was 10 ng of antigen per 100/tl (Fig. 3). No cross-reaction was detected with Vero cellular antigens (data not shown). The reactivity with other orbiviruses was not checked. Similar results were obtained when a rabbit hyperimmune IgG to AHS virus was used in the ELISA-DAS, as capture and labelled antibody (data not shown). Twenty eight samples of spleen from naturally ASHV infected equines and 31 samples from healthy animals were assayed using this combination of MAbs. Results are summarized in Table 1. There was agreement between ELISA values and AHSV isolation in tissue culture on Vero cells, except with one spleen which was doubtful in the ELISA test but positive in virus isolation. This sample was positive in the ELISA after passing once in Vero cells (data not shown). There were variations in absorbance in the ELISA test that could have resulted from differences in virus concentration in the spleen.
1.5
o
...\..\.
..
1,O'
05
I
0.I
0.01
ANTIGEN (~,g)
Fig. 3. Sensitivity of ELISA-DASby titration of AHSV antigen. Purified core antigen (---). Purified whole virus ( / / / / / ) . Purified bovine Rotavirus used as negativecontrol ( + + + ).
150
A.]. RANZ ET AL.
TABLE I Results of antigen detection by ELISA on spleen from healthy and AHSV infected equines No. of equines
ELISA values
27 I 31
TABLE 2
TOPOGRAPHICAL
median + SD
range
1.58+0.56 0.32 + 0.03 0.12+0.03
0.41 +2.00
~
!
~
~
e~ ~
SITES
ON THE AHSV V P 2
MABS
=-
~
e~ ~
i
~
~
~
~
8AE7 8CG1 tlHGIO 8t)H:J 6DF4 6DG2 6AF2 6AA3
+ i .. + + + -1
I
+ (~ + +
.. + +'/1
t.
AhlTIGENIC
SOS
NEUTRAUZING
I
I
YES
l
,/,,
YES
I+
+
.~ + +
+
II
YES
III
YES
IV
NO
V
YES
VI
NO
--
VII
YES
WEAK
/
+ : 4"
6AF3
+
4"
~tDF'
+
4-
j
E~i3B1
+
÷
+ + + + + 1
8BC2
+
+
+ + + + +
8BF2
÷
+
+ + + + +
8CE4
-k +
+ + + + +
8AE6
+
÷
+ + + + +
8DBI1
-P +
+ + + + +
8CBll
+
1" + + + + ÷
J ~
I(.!)7 Mabs showing
~
I 4 I
5I~MA
I
27 1 0
0.08__+0.21
DISTRIBUTION OF ANTIGENIC BIOTIhffLATED
PURIFIED
Virus isolation No. positive
i
WEAK
more than 70~, c o m p e t i t i o n
:Mab 6DF4 decreased viral i n f e c t i v i t y w h e n a seconO a n t i b o d y w a s a d d e d
Antigenic sites on VP2 We tested a panel o f MAbs in pairwise combinations in a competitive binding assay to identify distinct antigenic sites on AHSV VP2. Seven non overlapping antigenic sites (I to VII ) were defined and one area common to sites I and II (Table 2). Antigenic sites IV and VI were resistant to SDS denaturation. All the antigenic sites lacked neutralizing epitopes except antigenic sites V and VII, defined by MAbs SMAAB5 and 7CD7, respectively. MAbs 6DF4 exhibited neutralizing activity when a second antibody was added.
DIAGNOSTIC METHODS FOR AFRICAN HORSESICKNESS VIRUS
151
DISCUSSION
A panel of 32 MAbs recognizing two AHSV-4 structural proteins, VP2 and VP7, and one non-structural protein NS1 has been obtained. Three of the VP7-specific MAbs were used to develop a rapid test (ELISA-DAS) for the detection of AHSV antigen in the spleen of infected equines. This immunoassay could be done in 2.5 h and can detect 10 ng of purified AHSV cores in 100 /A. Two similar ELISAs have been described for the detection of AHSV antigen, using polyclonal antibodies (Du Plessis et al., 1990. Hamblin et al., 1991 ). The advantages of the test described in this manuscript are its specificity, inherent in the use of MAbs, and the speed of the assay. The use of MAbs that can be produced in large amounts with uniform quality will allow an easier standarization of this assay compared to those using polyclonal antibodies. The 3 MAbs used in this sandwich type ELISA have the same sensitivity as conventional polyclonal rabbit antibodies in their ability to detect antigen (Du Plessis et al., 1990), and have the additional advantage of being specific for the antigenic determinants present on AHSV VP7 which is a major protein of AHS virus and a putative group specific antigen, possibly with little variability between serotypes as occurs in Bluetongue virus, the prototype orbivirus (Huismans and Erasmus, 1981 ). This assay detected AHSV in spleen of infected animals with a sensitivity of 96.4%. Doubtful results should be confirmed by other techniques, like virus isolation in cell culture. The ELISADAS described in this report provides a valuable test to be used in routine diagnosis for rapid detection of AHSV in biological samples. During the replication of AHSV and other orbiviruses, tubular structures are synthesized in the infected cells (Huismans and Els, 1979). These structures are formed by NSI protein. We have obtained 8 MAbs binding to this protein, and all of them were able to stain tubules-like structures in AHSVinfected Vero cells 6 hours after infection in an immunofluorescence assay. This assay can be very useful as a confirmatory diagnostic test of AHSV infection. The antigenic structure of VP2 protein of AHSV was studied with 20 VP2 specific MAbs, by competition binding and neutralization assay. An understanding of the functional and antigenic properties of this viral surface protein is important for the development of a recombinant vaccine to control African horse sickness. VP2 has, at least, seven distinct antigenic sites and one bridge site. Only two of these sites (V and VII) were able to induce neutralizing antibodies. The low number of neutralizing MAbs (2 out 20) obtained could be due to the selection method used (indirect ELISA). Adsorption of proteins to plastic surfaces is known to affect the confirmation of certain antigenic determinants (McCullough et al., 1985; Bolwell et al., 1989). For that reason, MAbs specific to epitopes poorly exposed on the virus adsorbed to the well could be lost. In addition to these neutralizing MAbs, MAb
152
A.I. RANZ ET AL.
6 D F 4 also s h o w e d n e u t r a l i z i n g activity w h e n a s e c o n d a n t i b o d y , r a b b i t antim o u s e i m m u n o g l o b u l i n s , was a d d e d . T h i s result c o u l d be e x p l a i n e d if the e p i t o p e r e c o g n i z e d by M A b 6 D F 4 is highly r e p r e s e n t e d o n the viral surface. Alternatively, this e p i t o p e c o u l d be close to a n e u t r a l i z i n g one. In b o t h cases the d e p o s i t o f r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n s c o u l d e i t h e r block the rec e p t o r b i n d i n g site o r p r o m o t e a change in the virus s t r u c t u r e p r e v e n t i n g virus infectivity. Sites IV, r e p r e s e n t e d by 7 MAbs, a n d VI, d e f i n e d by M A b 8DA6, were resistant to SDS d e n a t u r a t i o n . T h e r e f o r e , it seems likely that antigenic sites o n the A H S V V P 2 p r o t e i n were p r e d o m i n a n t l y c o n f o r m a t i o n a l . T h i s i n f o r m a t i o n m i g h t be useful for the d e v e l o p m e n t o f a s u b u n i t vaccine for AHSV. ACKNOWLEDGEMENTS We wish to t h a n k H. H o o g h i n s , C. R u b i o a n d M.A. Cubillo o f N a t i o n a l A H S V R e f e r e n c e L a b o r a t o r y o f Algete, M a d r i d for p r o v i d i n g the A H S spleen samples, Dr. L. E n j u a n e s for t h e i r suggestions a n d Mrs. M a r i a del M a r Pitarch for t y p i n g the m a n u s c r i p t . T h i s w o r k was partially s u p p o r t e d by grant f r o m Spanish I n d u s t r y M i n i s t r y ( C D T I ) n u m b e r P-890282.
REFERENCES Bayer, E.A. and Wilchek, M., 1980. The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem. Anal., 26" 1-45. Bolwell, C., Brown, A.L. Barnet, P.V., Campbell, R.O. Clarke, B.E., Parry, N.R., Ovedridge, E.J., Brown, F. and Rowlands, D.J., 1989. Host cell selection of antigenic variants of footand-mouth disease virus. J. Gen. Virol., 70: 45-57. Bremer, C.W., 1976. A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus. Onderstepoort. J. Vet. Res., 43: 193-200. Du Plessis, D.H., Van Wyngaardt, W. and Bremer, C., 1990. An indirect sandwich ELISA utilising F(ab')2 fragment for the detection of African horsesickness virus. J. Virol. Methods., 29: 279-290. Hamblin, C., Met'tens, P.P.C., Mellor, P.S., Burroughs, J.N. Crowther, J.R., 1991. A serogroup specific ELISA for the detection and identification of AHS virus. J. Virol. Methods, 3 I: 285292. Hopkins, I.G., Hazrati, A. and Ozawa, Y., 1966. Development of plaque techniques for titration and neutralization tests with African horsesickness virus. Am. J. Vet. Res., 27: 96-105. Howell, P.G., 1962. The isolation and identification of further antigenic types of African horsesickness virus. Onderstepoort J. Vet. Res., 29:139-149. 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 serotypc-specific and group-specific antigens ofbluetongue virus. Onderstepoort J. Vet. Res., 48:51.58. Kearny, J.F., Radbruck, A, Liesegang, B. and Rajewsky, K., 1979. A new mouse myeloma line which has lost immunoglobulin expression but permits the construction of antibody secreting hybrid cell lines. J. lmmunol., 123:1548-1550.
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McCullough, K.C., Crowther, .I.R. and Butcher, R.N., 1985. Alteration in antibody reactivity with foot-and-mouth disease virus before and after binding to a solid phase or complening with specific antibody. J. lmmunol. Methods 82: 91-100. Melero, J.A. and Gonz~ilez Rodriguez, J., 1984. Preparation of monoclonal antibodies against glycoprotein Ilia of human platelets: Their effect on platelet aggregation. Eur. J. Biochem., 141: 421-427. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1987. Purification and properties of virus particles and cores of Bluetongue virus serotypes 1 and 4. Virology, 157: 375-386. Mirchamsy, H. and Taslimi, H., 1963. Adaptation of horsesickness virus to tissue culture. Nature (London), 198: 704-706. Nowinsky, R.C., Lostrom, M.E., Tam, M.R., Stone, M.R. and Burnette, W.N., 1979. The isolation of hybrid cell lines producing monoclonal antibodies against the p 15 (E) protein of murine leukemia viruses. Virology, 93:11 I-1126. Oellermann, R.A., 1970. Plaque formation by African horsesickness virus and characterization of its RNA. Onderstepoort. J. Vet. Res., 37 (2): 137-144. Oellermann, R.A., Els, H.J. and Erasmus, B.J., 1970. Characterization of African horsesickness virus. Arch Gesamte Virusforsch, 29:163-174. Ozawa, Y. and Hazrati, A., 1964. Growth of African horsesickness virus in monkey kidney cell cultures. Am. J. Vet. Res., 25:505-511. Sanz, A., Garcia-Barreno, B., Nogal, M.L., Vifiuela, E. and Enjuanes, L., 1985. Monoclonal antibodies specific for African swine fever virus proteins. J. Virol., 54:199-206. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Verwoerd, D.W., Huismans, H. and Erasmus, B.J., 1979. Orbiviruses. In: Comprehensive Virology, Vol 14 (Fraenkel-Conrat, H. and Wagner, R.R., eds), pp. 285-345. Plenum Publishing, New York.
143
Diagnostic methods for african horsesickness virus using monoclonal antibodies to structural and non-structural proteins Ana I. Ranz, Julita G. Miguet, Carmen Anaya, Angel Venteo, Elena Cort6s, Carmen Vela and Antonio Sanz Immunologia Y Genetica Aplicada S.A. (lngenasa) Madrid. Spain (Accepted 26 June 1992)
ABSTRACT Ranz, A.I., Miguet, J.G., Anaya, C., Venteo, A., Cort6s, E., Vela, C. and Sanz, A., 1992. Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins. Vet. Microbiol., 33: 143-153. A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the nonstructural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 ~1. The NS1specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.
INTRODUCTION
African horse sickness (AHS) is an arthropod-borne disease of Equidae, caused by a dsRNA orbivirus (AHSV) of the Reoviridae Family (Verwoerd et al, 1979). The infection of horses is characterized by a high mortality. The disease is endemic in central Africa, although outbreaks have occurred in North Africa and Southern Europe (Spain and Portugal) in recent years (1987-1991). Only one serotype, AHSV-4, has been isolated in Spain and Portugal during these outbreaks. The virus has a genome consisting of 10 segments of double-stranded RNA (Oellerman et al, 1970), each of which encodes at least one polypeptide. There are seven structural proteins, which form Correspondence to: A. Sanz, Immunologia y Genetica Aplicada S.A. (Ingenase-Hnos. Garcia Noblejas, 4 l-2°-28037-Madrid, Spain.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
144
A.I. RANZ ET AL.
a double-shelled virus particle. The outer capsid is composed ofthe two major proteins (VP2 and VP5), which are responsible for the viral neutralization and antigenic variability, whereas the inner capsid is composed of two major (VP3 and VP7), and three minor (VPI, VP4 and VP6) proteins. VP3 and VP7 are highly conserved among the nine AHSV serotypes (Oellerman et al., 1970; Bremer, 1976). AHSV also induces three non-structural proteins (NS 1, NS2 and NS3). A confirmed diagnosis of AHSV infection requires isolation and identification of the virus. This has usually been achieved by passing the putative virus in mouse brains (Howell, 1962) or cell cultures (Mirchamsy and Taslimi, 1963; Ozawa and Hazrati, 1964), and serological typing done by plaque reduction neutralization (Hopkins et al., 1966). However these methods are time consuming and require viable virus. The economic implications of outbreaks of AHSV make it essential to have rapid, sensitive and specific diagnostic tools. The availability of ASHV-specific monoclonal antibodies (MAbs) will be useful to develop diagnostic methods. In this paper we present the characterization of MAbs specific for AHSV-4 proteins, VP2, VP7 and NSI. A double antibody sandwich ELISA (ELISA-DAS) has been developed using VP7-specific MAbs, for the detection of AHSV in spleens from infected animals, as well as an immunofluorescence assay on AHSV-infected Vero cells using NSl-specific MAbs. In addition, these MAbs have been useful to study the antigenic structure of the outer capsid protein VP2. MATERIALS AND METHODS
Cells and virus Vero cells (ATCC) were grown in Dulbecco modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum ( FCS ). Hybridomas were grown in RPMI medium containing 15% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.4/IM aminopterine, 16 mM thymidine and antibiotics (penicillin and streptomycin). Cells were maintained in 8% CO2 atmosphere, The AHS virus serotype 4, used in this study, was isolated from the spleen of an infected horse and cloned twice by plaque isolation in Vero cells. Titres were determined by plaque assay on Vero monolayers as previously described (Oellermann, 1970). Virus was propagated in 850 cm 2 roller bottles of Vero cell cultures and purified as described for bluetongue virus by Mertens et al (1987). Production of hybridoma cell lines Hybridoma lines were obtained by fusion of spleen cells from ASHV immunized mice with the non-secreting myeloma cell line P3X63 Ag 8.653 (Kearny et al, 1979) following the procedure described by Nowinski et al.
DIAGNOSTIC METHODS FOR AFRICAN HORSESICKNESS VIRUS
145
( 1979 ) with slight modifications (Sanz et al. 1985 ). Screening of the hybrids was carried out by testing supernatants in an indirect ELISA using purified virus as antigen. MAbs were purified from ascitic fluid by HPLC (System Gold, Beckman ).
Immunoprecipitation and binding analysis The specificity of the MAbs was determined by two procedures, immunoprecipitation and binding of antigen to MAb-coated plates (immunoadsorption) as previously described (Melero and Gonz~ilez-Rodriguez, 1984; Sanz et al, 1985).
lm m unofluorescence studies Vero cells grown in micorslide culture chambers (Miles Scientific) were infected with ASH virus at multiplicity of infection (moi) 5. At different hours after infection, the cells were washed, fixed with methanol-acetone ( 1:1 ) and reacted with hybridoma supernatants containing the indicated MAb. After washing, cells were covered with fluoresceinated goat anti-mouse immunoglobulins (Sigma). Immunofluorescence was viewed with a Nikon Diaphot microscope equipped with epifluorescence
Immunoblotting analysis of MAbs AHS virus proteins from purified virions were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (Towbin et al, 1979). Membranes were saturated with 3% (w/v) non-fat dry milk in Tris buffered saline for 1 h, at room temperature. The reactivity of MAbs with polypeptides was detected with a biotin conjugated goat anti-mouse IGg (H + L) antibody (Sigma) and alkaline phosphatase-labelled streptavidin (Boehringer), using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro-blue tetrazolium ) as substrates.
Competitive binding ELISA Biotin (biotinamidocaproate N-hydroxysuccinande ester, Sigma) was conjugated as described (Bayer and Wilchek, 1980). MAbs were used in reciprocal competitive ELISA to eliminate the effects of antibody avidity on the results. 96-well plates were coated with 1/tg/well of purified virus, and then washed with phosphate buffered saline (PBS) containing 0.05% Tween 20. Competitions were performed with 10 fold dilutions of the purified MAb. The MAb was added to antigen-coated wells and allowed to react for 1 h at 37°C in the presence of a biotinylated MAb added at a predetermined optimal concentration. After adding streptavidin horseradish peroxidase-labelled (30 min, RT) and chromogen/substrate solution containing 40 mM 3-dimethyl-aminobenzoic acid (DMAB, Sigma), 0.78 mM 3-methyl-2-benzothiazolinone (MBTH, Sigma) and 0.003% H202 in phosphate buffer pH 7,
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A.L RANZ ET AL.
the absorbance of each well was measured in a micro ELISA autoreader (Flow) at 600 nm. Alternatively, o-phenylendiamine/H202 was used as chromogen/substrate and the absorbance measured at 450 nm.
Neutralization test Mabs alone or mixtures of MAbs were used in the neutralization test. Neutralization was determined by 90% plaque forming units (pfu) inhibition test. A virus dilution containing 100 pfu was mixed with various dilutions of MAbs (in presence or absence of rabbit anti-mouse immunoglobulins antibody, Sigma) and incubated overnight at 4°C plus 4 h at 37°C to increase the sentivity of the test. Virus adsorption to Vero cells in 24-well plates (Costar) was allowed to proceed at 37 °C for 2 h. The inoculum was removed and cells were covered with the agar overlay m e d i u m (DMEM, 1% agarose and 2% FCS). Five days after plates were fixed and stained with an aqueous alcoholic solution of crystal violet.
Double antibody sandwich Enzyme-Linked immunosorbent assay (ELISADAS) ELISA-DAS was used to test the ability of different MAbs to detect AHS virus in spleen extracts from infected horses. Briefly, wells were coated at various concentrations with different MAbs specific for AHS virus. One hundred/A of spleen extract, prepared by homogenizing 1 g of spleen in 10 ml of PBS, was added to the wells previously coated with antibody and incubated for 60 min at 37°C. U n b o u n d antigen was washed out. Bound antigen was detected by adding MAb conjugated to biotin and incubating 1 h at 37°C. The assay was developed by incubating with streptavidin-horseradish peroxidase and chromogen/substrate as described above. Reactions were considered positive when the OD values were three or more times greater than the mean of the OD obtained with negative samples. Absorbance values between 2 and 3 times greater than the mean of the negative samples were considered doubtful. RESULTS
Preparation and characterization of MAbs Thirty two hybridomas were derived from four fusion experiments. After cloning and stabilization MAbs were produced in ascitic fluid and further characterized by immunoprecipitation, immunoblotting, immunofluorescence staining of AHS virus-infected Vero cells, and virus neutralization. The reactivity of MAbs with purified AHSV virions and AHSV-infected cell lysate was determined by western blotting and immunoprecipitation respectively. Twenty MAbs reacted with VP2 protein, four MAbs recognized VP7 and eight MAbs reacted with NS 1. Figure 1 shows the proteins selected
DIAGNOSTICMETHODSFOR AFRICANHORSESICKNESSVIRUS
1
2
3
4
5
6
7
8
9
147
10 11
12
200 q 100 92.5
6,
~ ~
~ ~ '
'~
VP2
9 41D 6 NSl
4e
B ~
30
~
~
VP7
I
Fig. 1. Immunoprecipitation assay of 35S-methionine labelled AHSV proteins by MAbs. Line I, 14C-molecular weight markers. Lines 2 to 4 MAbs to VP2 (6DG2, 8AF8, 8DA6). Lines 5 to 7, MAbs to NS1 (5BB12, 5AF6, 6CA3). Lines 8 to 10 MAbs to VP7 (4AH9, 8DF4, 8DB6). Line 1 I MAb to rotavirus VP6 and line 12 mouse antiserum to AHSV.
by some MAbs from AHSV-infected cell lysate in immunoprecipitation assay. Polyclonal antibodies reacted with multiple AHS virus polypetides (lane 12 ). No reactivity was observed between MAbs with uninfected Vero cell lysate (data not shown). VP7-specific MAbs did not react in immunoblotting with the denatured protein. On the other hand eight VP2-specific reacted with denatured protein (data not shown). The removal of reducer agent did not restore the activity of non-reactive MAbs.
Reactivity of MAbs with intracellular antigens specified by AHS virus The reactivity of MAbs with AHSV-infected Vero cells was detected by immunofluorescence and is shown in Fig. 2. Three different fluorescence pat-
148
A.I. RANZ ET AL.
O
0
Fig. 2. lmmunofluorescence patterns of selected MAbs reacting with AHS virus-infected (A, B, C) and uninfected Vero cells (A', B', C'). A, A' Mab 8AF8 (VP2); B, B' Mab 8DB6 (VPT); C,C', Mab 5CGI0 ( N S I ) .
D I A G N O S T I C METHODS FOR AFRICAN HORSESICKNESS VIRUS
149
terns were observed: (i) diffuse cytoplasmic and perinuclear fluorescence; this pattern was obtained with the VP2-specific MAbs (Fig. 2A); (ii) fluorescence covering perinuclear and discrete cytoplasmic areas; this pattern was characteristic of the VP7-specific MAb (Fig. 2B); and (iii) staining of tubule-like structures; this pattern was characteristic of NS 1-specific MAbs (Fig. 2C) and was observed earlier than the others (at 6 hours after infection).
Detection of AHSV antigen in biological samples by ELISA-DAS Firstly, we investigated which combination of MAbs would be suited for a ELISA-DAS for the detection of antigen. Wells were coated with 1/tg of purified VP7-specific MAbs and, after incubating with purified antigen, combinations of biotin-labelled VP7-specific MAbs were added to each well. The highest sensitivity was observed when MAbs 4AH9 and 8DF4 were used as the capture antibodies and MAb 8DB6 as the labelled antibody. The sensitivity of the assay was 10 ng of antigen per 100/tl (Fig. 3). No cross-reaction was detected with Vero cellular antigens (data not shown). The reactivity with other orbiviruses was not checked. Similar results were obtained when a rabbit hyperimmune IgG to AHS virus was used in the ELISA-DAS, as capture and labelled antibody (data not shown). Twenty eight samples of spleen from naturally ASHV infected equines and 31 samples from healthy animals were assayed using this combination of MAbs. Results are summarized in Table 1. There was agreement between ELISA values and AHSV isolation in tissue culture on Vero cells, except with one spleen which was doubtful in the ELISA test but positive in virus isolation. This sample was positive in the ELISA after passing once in Vero cells (data not shown). There were variations in absorbance in the ELISA test that could have resulted from differences in virus concentration in the spleen.
1.5
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Fig. 3. Sensitivity of ELISA-DASby titration of AHSV antigen. Purified core antigen (---). Purified whole virus ( / / / / / ) . Purified bovine Rotavirus used as negativecontrol ( + + + ).
150
A.]. RANZ ET AL.
TABLE I Results of antigen detection by ELISA on spleen from healthy and AHSV infected equines No. of equines
ELISA values
27 I 31
TABLE 2
TOPOGRAPHICAL
median + SD
range
1.58+0.56 0.32 + 0.03 0.12+0.03
0.41 +2.00
~
!
~
~
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SITES
ON THE AHSV V P 2
MABS
=-
~
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i
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~
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8AE7 8CG1 tlHGIO 8t)H:J 6DF4 6DG2 6AF2 6AA3
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AhlTIGENIC
SOS
NEUTRAUZING
I
I
YES
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YES
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+
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+
II
YES
III
YES
IV
NO
V
YES
VI
NO
--
VII
YES
WEAK
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+
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+
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+
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+
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8CE4
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+ + + + +
8AE6
+
÷
+ + + + +
8DBI1
-P +
+ + + + +
8CBll
+
1" + + + + ÷
J ~
I(.!)7 Mabs showing
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I
27 1 0
0.08__+0.21
DISTRIBUTION OF ANTIGENIC BIOTIhffLATED
PURIFIED
Virus isolation No. positive
i
WEAK
more than 70~, c o m p e t i t i o n
:Mab 6DF4 decreased viral i n f e c t i v i t y w h e n a seconO a n t i b o d y w a s a d d e d
Antigenic sites on VP2 We tested a panel o f MAbs in pairwise combinations in a competitive binding assay to identify distinct antigenic sites on AHSV VP2. Seven non overlapping antigenic sites (I to VII ) were defined and one area common to sites I and II (Table 2). Antigenic sites IV and VI were resistant to SDS denaturation. All the antigenic sites lacked neutralizing epitopes except antigenic sites V and VII, defined by MAbs SMAAB5 and 7CD7, respectively. MAbs 6DF4 exhibited neutralizing activity when a second antibody was added.
DIAGNOSTIC METHODS FOR AFRICAN HORSESICKNESS VIRUS
151
DISCUSSION
A panel of 32 MAbs recognizing two AHSV-4 structural proteins, VP2 and VP7, and one non-structural protein NS1 has been obtained. Three of the VP7-specific MAbs were used to develop a rapid test (ELISA-DAS) for the detection of AHSV antigen in the spleen of infected equines. This immunoassay could be done in 2.5 h and can detect 10 ng of purified AHSV cores in 100 /A. Two similar ELISAs have been described for the detection of AHSV antigen, using polyclonal antibodies (Du Plessis et al., 1990. Hamblin et al., 1991 ). The advantages of the test described in this manuscript are its specificity, inherent in the use of MAbs, and the speed of the assay. The use of MAbs that can be produced in large amounts with uniform quality will allow an easier standarization of this assay compared to those using polyclonal antibodies. The 3 MAbs used in this sandwich type ELISA have the same sensitivity as conventional polyclonal rabbit antibodies in their ability to detect antigen (Du Plessis et al., 1990), and have the additional advantage of being specific for the antigenic determinants present on AHSV VP7 which is a major protein of AHS virus and a putative group specific antigen, possibly with little variability between serotypes as occurs in Bluetongue virus, the prototype orbivirus (Huismans and Erasmus, 1981 ). This assay detected AHSV in spleen of infected animals with a sensitivity of 96.4%. Doubtful results should be confirmed by other techniques, like virus isolation in cell culture. The ELISADAS described in this report provides a valuable test to be used in routine diagnosis for rapid detection of AHSV in biological samples. During the replication of AHSV and other orbiviruses, tubular structures are synthesized in the infected cells (Huismans and Els, 1979). These structures are formed by NSI protein. We have obtained 8 MAbs binding to this protein, and all of them were able to stain tubules-like structures in AHSVinfected Vero cells 6 hours after infection in an immunofluorescence assay. This assay can be very useful as a confirmatory diagnostic test of AHSV infection. The antigenic structure of VP2 protein of AHSV was studied with 20 VP2 specific MAbs, by competition binding and neutralization assay. An understanding of the functional and antigenic properties of this viral surface protein is important for the development of a recombinant vaccine to control African horse sickness. VP2 has, at least, seven distinct antigenic sites and one bridge site. Only two of these sites (V and VII) were able to induce neutralizing antibodies. The low number of neutralizing MAbs (2 out 20) obtained could be due to the selection method used (indirect ELISA). Adsorption of proteins to plastic surfaces is known to affect the confirmation of certain antigenic determinants (McCullough et al., 1985; Bolwell et al., 1989). For that reason, MAbs specific to epitopes poorly exposed on the virus adsorbed to the well could be lost. In addition to these neutralizing MAbs, MAb
152
A.I. RANZ ET AL.
6 D F 4 also s h o w e d n e u t r a l i z i n g activity w h e n a s e c o n d a n t i b o d y , r a b b i t antim o u s e i m m u n o g l o b u l i n s , was a d d e d . T h i s result c o u l d be e x p l a i n e d if the e p i t o p e r e c o g n i z e d by M A b 6 D F 4 is highly r e p r e s e n t e d o n the viral surface. Alternatively, this e p i t o p e c o u l d be close to a n e u t r a l i z i n g one. In b o t h cases the d e p o s i t o f r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n s c o u l d e i t h e r block the rec e p t o r b i n d i n g site o r p r o m o t e a change in the virus s t r u c t u r e p r e v e n t i n g virus infectivity. Sites IV, r e p r e s e n t e d by 7 MAbs, a n d VI, d e f i n e d by M A b 8DA6, were resistant to SDS d e n a t u r a t i o n . T h e r e f o r e , it seems likely that antigenic sites o n the A H S V V P 2 p r o t e i n were p r e d o m i n a n t l y c o n f o r m a t i o n a l . T h i s i n f o r m a t i o n m i g h t be useful for the d e v e l o p m e n t o f a s u b u n i t vaccine for AHSV. ACKNOWLEDGEMENTS We wish to t h a n k H. H o o g h i n s , C. R u b i o a n d M.A. Cubillo o f N a t i o n a l A H S V R e f e r e n c e L a b o r a t o r y o f Algete, M a d r i d for p r o v i d i n g the A H S spleen samples, Dr. L. E n j u a n e s for t h e i r suggestions a n d Mrs. M a r i a del M a r Pitarch for t y p i n g the m a n u s c r i p t . T h i s w o r k was partially s u p p o r t e d by grant f r o m Spanish I n d u s t r y M i n i s t r y ( C D T I ) n u m b e r P-890282.
REFERENCES Bayer, E.A. and Wilchek, M., 1980. The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem. Anal., 26" 1-45. Bolwell, C., Brown, A.L. Barnet, P.V., Campbell, R.O. Clarke, B.E., Parry, N.R., Ovedridge, E.J., Brown, F. and Rowlands, D.J., 1989. Host cell selection of antigenic variants of footand-mouth disease virus. J. Gen. Virol., 70: 45-57. Bremer, C.W., 1976. A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus. Onderstepoort. J. Vet. Res., 43: 193-200. Du Plessis, D.H., Van Wyngaardt, W. and Bremer, C., 1990. An indirect sandwich ELISA utilising F(ab')2 fragment for the detection of African horsesickness virus. J. Virol. Methods., 29: 279-290. Hamblin, C., Met'tens, P.P.C., Mellor, P.S., Burroughs, J.N. Crowther, J.R., 1991. A serogroup specific ELISA for the detection and identification of AHS virus. J. Virol. Methods, 3 I: 285292. Hopkins, I.G., Hazrati, A. and Ozawa, Y., 1966. Development of plaque techniques for titration and neutralization tests with African horsesickness virus. Am. J. Vet. Res., 27: 96-105. Howell, P.G., 1962. The isolation and identification of further antigenic types of African horsesickness virus. Onderstepoort J. Vet. Res., 29:139-149. 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 serotypc-specific and group-specific antigens ofbluetongue virus. Onderstepoort J. Vet. Res., 48:51.58. Kearny, J.F., Radbruck, A, Liesegang, B. and Rajewsky, K., 1979. A new mouse myeloma line which has lost immunoglobulin expression but permits the construction of antibody secreting hybrid cell lines. J. lmmunol., 123:1548-1550.
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McCullough, K.C., Crowther, .I.R. and Butcher, R.N., 1985. Alteration in antibody reactivity with foot-and-mouth disease virus before and after binding to a solid phase or complening with specific antibody. J. lmmunol. Methods 82: 91-100. Melero, J.A. and Gonz~ilez Rodriguez, J., 1984. Preparation of monoclonal antibodies against glycoprotein Ilia of human platelets: Their effect on platelet aggregation. Eur. J. Biochem., 141: 421-427. Mertens, P.P.C., Burroughs, J.N. and Anderson, J., 1987. Purification and properties of virus particles and cores of Bluetongue virus serotypes 1 and 4. Virology, 157: 375-386. Mirchamsy, H. and Taslimi, H., 1963. Adaptation of horsesickness virus to tissue culture. Nature (London), 198: 704-706. Nowinsky, R.C., Lostrom, M.E., Tam, M.R., Stone, M.R. and Burnette, W.N., 1979. The isolation of hybrid cell lines producing monoclonal antibodies against the p 15 (E) protein of murine leukemia viruses. Virology, 93:11 I-1126. Oellermann, R.A., 1970. Plaque formation by African horsesickness virus and characterization of its RNA. Onderstepoort. J. Vet. Res., 37 (2): 137-144. Oellermann, R.A., Els, H.J. and Erasmus, B.J., 1970. Characterization of African horsesickness virus. Arch Gesamte Virusforsch, 29:163-174. Ozawa, Y. and Hazrati, A., 1964. Growth of African horsesickness virus in monkey kidney cell cultures. Am. J. Vet. Res., 25:505-511. Sanz, A., Garcia-Barreno, B., Nogal, M.L., Vifiuela, E. and Enjuanes, L., 1985. Monoclonal antibodies specific for African swine fever virus proteins. J. Virol., 54:199-206. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Verwoerd, D.W., Huismans, H. and Erasmus, B.J., 1979. Orbiviruses. In: Comprehensive Virology, Vol 14 (Fraenkel-Conrat, H. and Wagner, R.R., eds), pp. 285-345. Plenum Publishing, New York.
