Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting

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1 Journal of General Virology (1993), 74, Printed in Great Britain 81 Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting M. D. Laviada,* M. Arias and J. M. S~inchez-Vizcaino Departamento de Sanidad Animal, CIT-INIA, Embajadores no. 68, Madrid, Spain The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The crossreactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes. Introduction African horsesickness is a virus disease of Equidae, causing high mortality in horses. The virus is transmitted by arthropods of the genus Culicoides (Du Toit, 1944; Mellor et al., 1990). The disease is enzootic in sub- Saharan Africa, although occasional severe epizootic episodes have occurred in North Africa, the Middle East and in certain European countries (McIntosch, 1958). In 1987 an outbreak took place in Spain, and in the following years (1988 to 1990) several severe outbreaks were reported in Spain, Morocco and Portugal. African horsesickness virus (AHSV) is classified in the Orbivirus genus of the family Reoviridae (Borden et al., 1971; Murphy et al., 1971). Nine different serotypes of the virus have been identified in Africa (McIntosch, 1958; Howell, 1962) but only one serotype, AHSV-4, has been isolated in Spain and Portugal since 1987 (Yubero, 1988a, b). Little information is available about AHSV proteins and their genetics. Electron microscopic evidence and physicochemical studies indicate a close morphological and biochemical relationship with bluetongue virus (BTV), the prototype orbivirus (Oellermann et al., 1970; Bremer, 1976). Like BTV, AHSV consists of 10 ds-rna segments and seven structural proteins forming a doubleshelled virion. One structural protein, VP7, has been sequenced (Roy et al., 1991). At least two non-structural proteins, NS1 and NS2, have been identified in infected cells (Devaney et al., 1988; Huismans & Els, 1979; van Staden et al., 1991) and two more, NS3 and NS3a, have been identified by in vitro translation from the smallest RNA segment (van Staden & Huismans, 1991). Recently, Grubman & Lewis (1992) have found three major (NS1, NS2 and NS3) and variable amounts of two minor (NS4 and NS4a) non-structural proteins in AHSV-4-infected cells. NS4 and NS4a are equivalent to NS3 and NS3a of van Staden & Huismans (1991) because both are encoded by the smallest RNA segment (segment 10). Grubman & Lewis (1992) have also determined the coding assignments of all the purified genome segments by in vitro translation. The genetic relatedness of dsrna segments from all nine AHSV serotypes has been investigated using cloned genome segments of AHSV-3 as probes in Northern blot hybridization experiments (Bremer et al., 1990). However, the degree of cross-reactivity between the proteins of the nine different AHSV serotypes has not been reported. We report the characterization of the proteins of AHSV-4 and the identification of proteins that elicit the major antibody responses in both vaccinated and naturally or experimentally infected horses. In addition, we have analysed the cross-reactivity between AHSV-4 proteins and sera obtained from horses experimentally infected with eight different AHSV serotypes. This information will be relevant for the future development of vaccines and new diagnostic procedures, particularly to distinguish between vaccinated and naturally infected animals. Finally, these studies have allowed the adaptation of SGM

2 82 M. D. Laviada, M. Arias and J. M. Sdnchez-Vizcalno (a) 0 1 (b) K- 200K - VP1 VP2 92K - - VP3 69K-- VP5 P58 92K- ~ ~ ~ VP3 6 9 K - ~,~ VP P58 46K-- VP6 P K-, ~ 9 :...!i!]i; i; i~i,2,~o~o; VP6 P48 VP7 ~,? VP7 30K-,~o P28 " ~ ' - P23 ~ Fig. 1. Identification of AHSV-induced polypeptides in infected Veto cells labelled at different times (h) p.i. as indicated. (a) Infected with an m.o.i, of 0.1 and (b) an m.o.i, of 10. Each virus-induced polypeptide is marked with an arrow. a Western immunoblotting assay for AHSV antibody detection, which could be useful as an alternative to currently accepted methods of serological diagnosis. Methods.~.--- P21 P20 Fig. 2. Induced and structural proteins of AHSV in Vero cells. Lane 1, mock-infected Vero cells; lane 2, AHSV-induced polypeptides in Vero cells at 48 h p.i. ; lane 3, purified AHSV labelled with [3SS]methionine; lane 4, purified AHSV stained with Coomassie blue; lane 5, immunoprecipitation of purified AHSV with a serum from a naturally AHSV-4-infected horse. with [3SS]methionine for 2 h at different times from 8 to 56 h postinfection (p.i.). Mock-infected cells were radiolabelled as controls. Lysates of mock-infected and infected cells were analysed by SDS-PAGE. Radiolabelled, purified virus was prepared by infecting cells in a 25 cm 2 culture flask at a multiplicity of When the first signs of c.p.e, were observed (at 2 days p.i.), cells were labelled with 500 pci [ass]methionine in 1 ml of medium without methionine and 2 % foetal calf serum for 24 h. Cells were harvested, mixed with unlabelled infected cells from 20 roller bottles and purified as described (Laviada et al., 1992). Viruses, cells andantisera. A Spanish strain of AHSV isolated in 1989 from an infected horse was grown in Vero cells as described (Laviada et al., 1992) and used after three to seven passages. Several groups of sera were used. One serum from an experimentally AHSV-4-infected horse was kindly provided by Dr C. Mebus (Plum Island Disease Control Center, U.S.A.), as were sera from horses vaccinated with attenuated serotypes 1, 2, 3, 4, 6, 7, 8 and 9. Eleven sera from horses vaccinated with a live attenuated AHSV-4 vaccine and two sera from horses vaccinated with a polyvalent (serotypes 1 to 7 and 9) live attenuated vaccine (Ozawa et al., 1965) supplied by the Veterinary Research Institute, Onderstepoort, South Africa. One serum from a naturally AHSV-4-infected horse was provided by Dr H. Hooghuis (National AHSV Reference Laboratory, Algete, Madrid, Spain). Two groups of 11 sera from horses vaccinated with the inactivated purified vaccine from Rh6ne-M~rieux (Dubourget et al., 1991) and the live attenuated AHSV-4 vaccine from South Africa, respectively, were kindly provided by Dr M. Lombard (Rh6ne-M~rieux, France). One equine serum prepared in 1984 was used as a negative control. S D S - P A G E. This was carried out on 15% acrylamiden,n'-diallyltartardiamide (DATD) gels (Escribano & Tabards, 1987). After electrophoresis, gels containing samples from radioimmunoprecipitation assays were fluorographed following the method described by Bonner & Laskey (1974) Infection andlabelling o f cells. Vero cells were infected at low and high multiplicity (0.1 and 10 respectively) with AHSV-4 and radiolabelled Immunoblotting assay. The binding of antibodies to viral proteins separated by electrophoresis and transferred to nitrocellulose paper Radioimmunoprecipitation. Radiolabelled cytoplasmic extracts from infected and mock-infected Veto cells were prepared as described previously (Laviada et al., 1992). The radioimmunoprecipitation assay was performed as described (Alcaraz et al., 1990) except that Streptococcus Protein G (Calbiochem) was used instead of Staphylococcus Protein A to obtain binding to horse Igs.

3 African horsesickness virus proteins vp1 - vp2 - vp3 ~ ~ ~ - VP2 - vp5 - P58 - vp6 - P48 - vp7 m... VP5 ~" ~ - P58... P48 - P46 - P32 ~ -... P23 _ e21 ~.~ - P20 :~ - P23 : ~,: ~. ~ - P21 ~!~: ~ - P20 iv ii: Fig. 3. Antigenicity of AHSV-induced polypeptides determined by immunopreclpitation. The labelled AHSV-infected Vero cell lysates were immunoprecipitated using antisera from a experimentally AHSV-4-infected horse (lane 5), and monovalent (serotype 4) vaccinated horses (lanes 6 to 11). A negative serum (lane 3) and a mock-infected Vero cell lysate immunoprecipitated with the same serum as in lane 5 (lane 4) were used as negative controls. was performed by the method of Towbin et al. (1979) with minor modifications. AHSV proteins, semi-purified as described by House et al. (1990), were separated by SDS-PAGE in 15 % acrylamide DATD gels (Escribano & Tabards, 1987). Separated proteins were transferred to a nitrocellulose membrane filter at a constant current of 280 ma for 6 h at 4 C. Immunoblotting of nitrocellulose membrane cut into strips was carried out as described (Escribano et al., 1990), using the sera at a 1:20 dilution and peroxidase-conjugated rabbit anti-horse Ig at a 1 : 500 dilution, and an incubation time of 1 h at 37 C. The bands recognized by the sera were developed by the 4-chloro-l-naphthol technique (Hawkes et al., 1982). Results Virus-induced polypeptides To identify virus-induced polypeptides, mock- and AHSV-4-infected Vero cells were pulse labelled with [35S]methionine and the cell lysates were analysed by SDS-PAGE. For radiolabelling, infection was carried out at different times p.i. (0, 8, 24, 32, 48 and 56 h p.i.) at both low and high multiplicity (0.1 and 10 respectively). Twenty-two polypeptides with Mrs of between 120K and 20K could be detected by comparison of infected and mock-infected Vero cell polypeptides (Fig. 1, marked with arrows). Preparations of purified 35S-labelled AHSV were obtained by sedimentation through a 4 to 40 % sucrose gradient. The purified particles were analysed by SDS-PAGE (Fig. 2, lanes 3 and 4). Four major and two minor structural polypeptides were observed and designated VP2 (130K), VP3 (90K), VP5 (63K), VP7 (38K), VP1 (120K) and VP6 (52K) respectively. One minor protein, VP4, was visible only on some gels as a very faint band, the position of which is marked with a dotted line in Fig. 2. It should be noted that VP5 was resolved as a doublet, clearly visible in purified virus preparations. Both proteins of the doublet reacted with two monoclonal antibodies specific for VP5 (data not shown). In addition, at least four major non-structural proteins (P58, P48, P23 and P21) were detected by comparison of infected cell extracts and purified virions (Fig. 2, lane 2). Virus polypeptides detected by using AHSV antibody Evidence that 11 of the 22 virus-induced proteins in infected cells are virus-coded was based on their recognition by antisera raised against AHSV. Infected and non-infected 3~S-labelled cell extracts were immuno-

4 i 84 M. D. Laviada, M. Arias and J. M. S6nchez-Vizca[no precipitated with different sera obtained from AHSV-4- infected and vaccinated horses (Fig. 3). All seven sera reacted with the two major structural proteins VP2 and VP7, five sera reacted with P21, four with P48 and two with P58. As shown in Fig. 3, the immunoprecipitation reaction was specific and allowed the identification of two more minor virus-coded proteins, P46 and P20, that were difficult to detect without immunoprecipitation. When different hyperimmune sera were analysed, at least 13 polypeptides could be identified by immunoprecipitation, including a group of proteins with an Mr lower than 30K (Fig. 3, lanes 9 to 11). Similarly, we confirmed the antigenicity of the structural proteins using a serum from a naturally AHSV-4-infected horse to precipitate purified 35S-labelled virus preparations (Fig. 2, lane 5). The results indicated that viral proteins VP2, VP7, P48, P21 and P20 are the most antigenic. To investigate whether AHSV-4 proteins are related to those of the other serotypes of AHSV a radioimmunoprecipitation assay was carried out using AHSV- 4 proteins and sera from horses immunized with serotypes 1, 2, 3, 6, 7, 8 and 9. The results of these crossreactivity experiments showed that the outer capsid protein VP2 exhibits major antigenic variation among the various AHSV serotypes, whereas viral protein VP7 is highly conserved. Other virus-coded proteins, e.g. VP5, P58, P48, P21 and P20, were only precipitated by some heterologous antisera indicating different levels of cross-reactivity (Fig. 4). Immunoblotting of AHS V-4 proteins Nitrocellulose strips containing SDS-PAGE-resolved AHSV-4 proteins were incubated with a collection of sera from polyvalent and monovalent attenuated AHSV- 4 vaccinated horses. Viral proteins VP5, P58, P48, P46, P21 and P20 showed the strongest reaction (Fig. 5). VP2, VP3, VP6, VP7 and P28 could also be detected using hyperimmune sera (Fig. 5, lanes 19 and 21). The lack of reactivity of VP7 in many immunoblots despite the strong reactivity of this protein in the radioimmunoprecipitation assay is surprising. No differences were observed between the reactivity of sera from vaccinated (attenuated vaccine) and infected horses (data not shown). When the cross-reactivities were compared using sera from horses immunized with seven different serotypes, the results demonstrated that viral proteins VP5, P48, P46, P21 and P20 are highly conserved among these eight serotypes. In contrast and as expected, VP2 showed the greatest variation (Fig. 5, lanes 2 to 8). To establish differences between horses vaccinated with live, attenuated virus and those vaccinated with inactivated virus (purified virions), AHSV-4 structural 1 2 b VP1 - VP2 ~ -VP2 - VP3... VP3 - VP5, -VP5 -P58 ~ ~ P58 VP6 - P48 ~,~ ~ ~ ~ - P48 -VP7 --~ ".... ~L77.~7... VP7 - P28,~ - P28 - P23 -_ P21 P20 ~ i - P20 P21 Fig. 4. Cross-reactivity of AHSV-4 proteins with sera from horses immunized with one of eight different serotypes (attenuated vaccine) determined by immnnoprecipitation. The 35S-labelled AHSV-infected Vero cell lysate shown in lane 2 was precipitated with a negative serum (lane 3) or with immune sera against serotypes 1, 2, 3, 4, 6, 9, 8, 9 and 7 (lanes 4 to 11, respectively) el.... :=" "=- "i ~ ~ -~- ',m-vp3 D. ~:,w.:.~ i;. VP5 ~:~ i....!li P48 VP7 -- Fig. 5. Western immunoblotting of AHSV-induced polypeptides with sera from horses immunized with serotypes 1, 2, 3, 6, 9, 7, 8 and 9 (lanes 2 to 8, respectively), from horses immunized with polyvalent vaccine (serotypes 1 to 7 and 9; lanes 9 and 10) and horses immunized with a monovalent (type 4) vaccine (lanes 11 to 21). A negative serum (lane 1) was used as a control. and non-structural proteins were tested by immunoblot analysis using sera collected from both groups of horses. As shown in Fig. 6, all sera had similar patterns of reactivity with structural proteins and non-structural proteins P58, P48 and P46. However, P21 and P20 could not be detected in sera from horses vaccinated with the inactivated vaccine.

5 African horsesickness virus proteins ~) VP2 VP3 - VP5 P58 VP6 P48 VP7 - P21 P20- (b) i i VP2 VP3 - VP5 P58 VP6 P48 VP7 - P21 P20 - of immunoblotting pattern of sera from horses vaccinated with an attenuated AHSV-4 F i g. 6. C o m p a r i s o n v a c c i n e (a) a n d t h a t o f s e r a from horses vaccinated with an inactivated purified AHSV-4 vaccine (b). Discussion In the present study we have identified the AHSV-4 proteins and elucidated their antigenic properties. 85 Analysis of 3~S-labelled AHSV-infected Vero cells by PAGE showed at least 22 virus-induced polypeptides, seven of which were identified as structural polypeptides by comparison with polypeptides from 35S-labelled purified virus preparations (Fig. 2). VP5 was resolved as a doublet by electrophoresis of purified virus and by immunoblotting when a semi-purified virus preparation was used as antigen. However, Grubman & Lewis (1992) found only a single VP5 band in preparations of AHSV-4. This difference could be due to the different gel system used, but further investigation will be needed to elucidate this. VP4 is a minor protein which is difficult to identify in purified virus preparations; Grubman & Lewis (1992) have also shown this. The major non-structural proteins, defined as P58 and P48, correspond to those previously identified as NS1 and NS2 in AHSV (Devaney et al., 1988; Huismans & Els, 1979), based on their relative Mrs and molar ratios. However the relative position of these proteins in the electrophoretic pattern, in respect to VP5 and VP6, showed some differences to the pattern obtained with serotype 3 (Devaney et al., 1988). However, P58 is synthesized in a large excess over the other proteins and migrates as a broad, slightly diffuse band, which is similar to that described for BTV NS1 (van Dijk & Huismans, 1988). P48 has an M r similar to that estimated from the sequence of the NS2-encoding genome segment 8 of AHSV-9 (van Staden et al., 1991), allowing for posttranslational phosphorylation (Devaney et al., 1988). The pattern of proteins reported here is in agreement with that of serotype 4 recently described by Grubman & Lewis (1992). Two AHSV non-structural proteins, NS3 and NS3a, have been identified recently by in vitro translation (van Staden & Huismans, 1991) from the smallest dsrna segment, S10; NS3a is a truncated form of NS3. These proteins have been estimated to be of M r 24K and 23K. Grubman & Lewis (1992) have also described the presence of two non-structural proteins, NS4 and NS4a, in AHSV-4-infected cells. These have M~.s of 26K and 25K, respectively, and are encoded by genome segment 10. These proteins reacted with a convalescent serum. We have characterized at least six virus-induced proteins with an M r lower than 30K, of which P20 and P21 could correspond to NS4 and NS4a because they have been demonstrated to be the most immunogenic proteins and to react strongly with sera from vaccinated and infected horses. These proteins also reacted with sera from horses infected with other AHSV serotypes. A lesser degree of cross-hybridization between dsrna $10 of various AHSV serotypes than between serotypes of BTV has been described (Ritter & Roy, 1988; Bremer et al., 1990). However, comparison of the amino acid sequences of AHSV-3 and AHSV-9 NS3 has identified two regions of

6 86 M. D. Laviada, M. Arias and J. M. Sdnchez- Vizcaino approximately 45 amino acids that display 98 % similarity (van Staden & Huismans, 1991). These might be the antigenic areas reactive in the serotypes studied here. P23 could correspond to NS3 described by Grubman & Lewis (1992) because both are major non-structural proteins that migrate near to NS4 and NS4a. Eleven of the AHSV-induced proteins found in infected cells have not been identified as the products of specific genes. These proteins should be investigated further. From our studies we conclude that at least I 1 antigens can induce antibodies in infected horses (Fig. 3 and 5). The major antigenic structural proteins were VP2 and VP7 in radioimmunoprecipitation assays and VP5 in Western immunoblotting. The most reactive non-structural proteins in both tests were P58, P48, P21 and P20. Some differences have been observed in the AHSV-4 proteins that react with specific antisera in radioimmunoprecipitation and Western immunoblotting assays. Thus, the most immunogenic epitope in VP7 seems to be conformational, since it is recognized in the radioprecipitation assay but only weakly when the protein is denatured in immunoblotting. The presence of one antigenically dominant conformational epitope in this protein has been suggested previously (Laviada et al., 1992). A similar observation was also made for VP2. In contrast, the immunogenic epitopes of VP5, P58, P48, P46, P21 and P20 are located in the primary structure. It is noteworthy that responses to P20 and P21 always occurred together and were usually stronger than responses to non-structural proteins P58 and P48 (NS1 and NS2). These proteins may be developed as diagnostic reagents for distinguishing infected horses (or horses vaccinated with attenuated vaccines) from those vaccinated with inactivated purified virus vaccines (Dubourget et al., 1991). Our preliminary studies with a group of sera raised against both types of vaccines (Fig. 6) support this view, but an extensive number of sera will have to be examined to confirm these data. Radioimmunoprecipitation and Western immunoblotting assays revealed that the outer capsid protein VP2 varied among the different AHSV serotypes, as would be expected for a protein involved in serotype differentiation. This would be the equivalent of the BTV outer capsid protein VP2 and is in agreement with the experiments carried out by Bremer et al. (1990) in which a segment 2 clone hybridized only to the genome of the homologous serotype under high stringency conditions. Structural proteins VP3, VP5 and VP7 as well as nonstructural proteins P48, P46 and the doublet P2 l-p20 are highly conserved among all serotypes. We thank Dr M. Lombard, Dr H. Hooghuis, and Dr C. Mebus for providing the different sera, J. Bustos for her excellent technical assistance and I. Loma for typing the manuscript. We are also very grateful to Dr P. Roy for reviewing this manuscript. This work was supported partly by INIA grant no and CDTl/Ingenasa grant no References ALCARAZ, C., DE DIEGO, M., PASTOR, M. J. & ESCRIBANO, J. A. (1990). Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus. Journal of Veterinary Diagnostic Investigation 2, BONNER, W. M. & LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyaerylamide gels. European Journal of Biochemistry 46, BORDEN, E. C., SHOPE, R. E. & MURPHY, F. A. (1971). 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