Cloning, characterization and expression of the ... - Semantic Scholar
Journal of General Virology (1994), 75, 3629 3633. Printed in Great Britain
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Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3 F. T. V r e e d e t and H . H u i s m a n s * Department of Genetics, University of Pretoria, Pretoria 0001, South Africa
The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50-5 % identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35 % identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydro-
philicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculovirus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.
African horsesickness (AHS) is a severe, often fatal, viral disease of Equidae endemic to sub-Saharan Africa. The aetiological agent, African horsesickness virus (AHSV), is transmitted by arthropods of the genus Culicoides. AHSV is classified together with a number of other double-stranded RNA viruses, such as bluetongue virus (BTV), in the genus Orbivirus of the family Reoviridae (Holmes, 1991). Nine different AHSV serotypes have been identified with little, if any, cross-neutralization between them (McIntosh, 1958; Howell, 1962). Like BTV, the AHSV particle consists of a doublelayered capsid containing the viral genome, which is composed of 10 dsRNA segments encoding seven structural (VP1 to VP7) and at least three non-structural proteins (Grubman & Lewis, 1992). The outer capsid layer is composed of VP2 and VP5, and surrounds an icosahedral core particle which is composed primarily of VP3 and VP7, with lesser amounts of VP 1, VP4 and VP6 (Bremer, 1976; Bremer et al., 1990; Laviada et al., 1993). Studies with BTV have shown that VP2 is highly variable and is the main determinant of serotype specificity and the neutralization-specific immune response (Huismans
& Erasmus, 1981). VP2 recovered from purified BTV (Huismans et al., 1987) or derived from baculovirus expression vectors (Roy et al., 1990) has been demonstrated to induce neutralizing antibodies and to elicit protection in sheep against virulent viral challenge. VP5 was further found to enhance the neutralization (and protective) immune response (Roy et al., 1990), and recently considerable effort has been exerted in the evaluation of virus-like and core-like particles as subunit vaccines against BTV (Van Dijk, 1993). It has recently been demonstrated that neutralizing epitopes for AHSV are located on VP2 and that antibodies to these epitopes are protective in a neonatal mouse model (Burrage et al., 1993). This suggests that recombinant VP2 bearing these neutralizing epitopes may have efficacy as a subunit vaccine against AHSV. In vitro translation studies have shown that AHSV VP2 is encoded by genome segment 2 (Grubman & Lewis, 1992). The entire sequence of this segment of AHSV-4 has been determined and the deduced VP2 amino acid sequence compared with the cognate proteins of epizootic haemorrhagic disease virus (EHDV) and BTV (Iwata et al., 1992). In this paper, we describe the cloning and sequencing of the VP2 gene of AHSV-3, presenting the first opportunity to qualitatively compare the outer capsid protein gene of two AHSV serotypes and enabling the identification of regions of possible antigenic importance.
t Presentaddress: BiochemistryDivision,OnderstepoortVeterinary Institute, Onderstepoort 0110, South Africa. The sequencedata reported in this paper have been registeredwith the GenBank Data Libraryunder the accessionnumber U01832. 0001-2550 © 1994SGM
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Fig. 1. Best-fit alignments of the segment 2 non-coding nucleotide sequences (lower case) and the VP2 amino acid sequences (upper case) of AHSV-3 (top) and AHSV-4 (bottom). Conserved residues are indicated by asterisks (identical) or dots (similar).
In addition, we have expressed VP2 of AHSV-3 in a baculovirus expression system and present the results of initial studies of the ability of recombinant AHSV-3 VP2 to induce a neutralization-specific immune response. The strategy employed for the preparation and cloning of cDNA from AHSV-3 segment 2 dsRNA was a modification of the method of Cashdollar et al. (1984) as described by Bremer et al. (1990). The clones were analysed by PstI digestion and hybridization to a Northern blot of AHSV-3 dsRNA. Four overlapping partial cDNA clones (1200, 2000, 1500 and 800 bp), representing the entire AHSV-3 segment 2, were obtained and sequenced. The non-coding nucleotide sequences along with the deduced amino acid sequence of the coding region are shown in Fig. 1 in a direct comparison with VP2 of AHSV-4. Genome segment 2 of AHSV-3 was determined to be 3221 bp in length with a calculated base composition of 14-93% C, 26.12% G, 31.32% A and 27.63 % U. The 5'- and T-terminal hexanucleotides of the AHSV-3 genome segment 2 were 5' GUUUAA and ACUUAC 3' respectively, which supports the consensus AHSV terminal sequences recently proposed by Mizukoshi et al. (1993) that are 5' GUUuA~,A a and cACAUACt,3', based on previous studies involving sequence analyses of the viral RNA species of AHSV. The longest open reading frame (ORF) identified was defined by an AUG at position 13 to 15 and a TAA at position 3184 to 3186, delineating 5' and 3' non-coding regions of 12 bp and 35 bp respectively. The flanking sequences of the AUG codon, CACCAUGG, are comparable to the consensus
sequence for initiation of translation (GCCGCCACCAUGG) identified by Kozak (1987). The AHSV-3 segment 2 gene was deduced to encode a protein composed of 1057 amino acids with a calculated Mr of 123078K. Comparisons of the VP2 genes of AHSV-3 and AHSV4 revealed a 58"7% conservation of nucleotides in type and position, which translates to a 50.5% identity of amino acids in type and position in a best-fit alignment requiring six gaps involving 11 amino acids. When amino acids of similar character were taken into consideration, the similarity index was 71-3 %. The regions that exhibit the greatest similarities are located near the termini and in the centre of the protein (Fig. 2 a). This was confirmed by Diagon analyses (results not shown). These findings correlate with data from extensive comparisons of VP2 of various BTV serotypes which yielded similar percentages and patterns of amino acid conservation (Fukusho et al., 1987; Ghiasi et al., 1987; Roy, 1989; Yamaguchi et al., 1988). Possible functional and conformational roles for the carboxy-terminal and central conserved regions have been suggested. Approximately one-third of the amino acids of the VP2 proteins of both AHSV-3 and AHSV-4 are charged. The hydropathicity profiles of AHSV-3 and AHSV-4 VP2 (Fig. 2b) demonstrated significant similarity, revealing strong hydrophilic characters with a few regions of hydrophobicity evident particularly toward the C terminus. The variability associated with the distinctively hydrophilic domain between amino acids 128 and 309 suggests that the
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determinant(s) of the serotype-specific immune response on VP2 may be located in this region within the overall structurally conserved antigen. In order to study the immunogenicity of VP2 of AHSV-3, an Autographa californica nuclear polyhedrosis virus (AcNPV) recombinant containing the full-length clone of the AHSV-3 VP2 gene under control of the p 10 promoter (Vlak et al., 1988) was constructed. A single 3"2 kb product, representing AHSV-3 segment 2, was obtained by PCR amplification of an alkaline sucrose gradient-purified fraction of large AHSV-3 cDNA with synthetic oligonucleotide segment 2 termini-specific primers as described in Nel & Huismans (1990). The primers were designed to include BglII restriction sites to facilitate cloning of the segment 2 gene into the
Fig. 3. Autoradiograph (a) and Western blot (b) of 10% SDS-PAGEseparated extracts of mock-infected (lanes 1) and AHSV-3-infected (lanes 2) Vero cells, AHSV-3 segment 2 recombinant baculovirus (lanes 3), wild-type AcRP23-lacZ (lanes 4) and mock-infected (lanes 5) S. frugiperda ceils. The putative recombinant baculovirus-expressed VP2 protein is marked with an arrow.
baculovirus dual transfer vector pAcUW3 (Weyer & Possee, 1991). Following verification of the correct orientation of the VP2 gene in the transfer vector relative to the baculovirus pl0 promoter, limited sequencing of the termini confirmed the full-length status of this clone. AcNPV VP2 recombinants were obtained by cotransfection of Sf9 cells with the AHSV-3 segment 2 recombinant transfer vector DNA and linearized AcRP23-1acZ DNA, a lacZ recombinant AcNPV derived by Possee & Howard (1987). Putative recombinant viruses that exhibited a white plaque phenotype following exposure to X-gal, were isolated by two rounds of plaque purification. VP2-specific recombinant baculoviruses were identified by dot hybridization of recombinant viral DNA to an AHSV-3 segment 2-specific probe. Using in situ hybridization analyses (Paeratakul et al., 1988), VP2-specific mRNA was detected between 18 h post-infection (p.i.) and 40 h p.i. (results not shown). In order to determine whether AHSV-3 VP2 was synthesized in cells infected with the recombinant AcNPV, the cells were pulse-labelled with [35S]methionine between 28 h and 31 h p.i. and the labelled proteins were analysed by SDS-PAGE (Fig. 3 a). A protein o f M r approximately 116K that comigrated with VP2 from AHSV-3-infected cells, was shown to be synthesized in recombinant AcNPV-infected cells but was absent in mock- or wildtype AcRP23-1acZ-infected cells. In the latter case, the M r l I6K fl-galactosidase protein was expressed and migrated as a band of approximately 120K in SDSPAGE. The band in lane 3 corresponding in apparent M,. to the fl-galactosidase band in lane 4, was identified as a non-specific protein produced in baculovirus-infected cells. Confirmation of the viral origin of the putative VP2
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Short communication
protein was obtained by Western immunoblotting of the SDS-PAGE-separated proteins (Fig. 3b). A specific reaction of VP2 with rabbit anti-AHSV-3 serum was observed whereas no interserotype cross-reactivity with horse anti-AHSV-4 serum was exhibited (results not shown). The band of M r approximately 110K in lane 3 which also reacted with the antiserum may represent a truncated form of VP2. These results indicated that AHSV-3 VP2 was expressed during the recombinant virus infection. The yield of expressed VP2 in recombinant AcNPV-infected cells was estimated to be 0'5 to 1 lag/106 cells. This was significantly less than expected, being at least threefold lower than the yield of baculovirus-expressed BTV VP2 (Inumaru & Roy, 1987). The reason for the low yield of AHSV-3 VP2 is currently under investigation. To assess the ability of baculovirus-expressed VP2 to induce a neutralization-specific immune response, plaque-reduction assays were conducted as described by Huismans & Erasmus (1981) with antibodies raised in rabbits and guinea-pigs to recombinant baculovirusinfected cell lysates. The animals were injected intramuscularly with VP2-containing lysates (approximately 60 lag VP2/ml) suspended in an equal volume of Montanide Incomplete Seppic Adjuvant (ISA50). The guinea-pigs each received an initial inoculation of 15 lag VP2 on day 0 followed from day 28 post-inoculation with weekly boosters of 6 to 9 lag VP2. The rabbits were given an initial dose of 30 lag UP2 followed at days 21 and 54 post-inoculation with a further 12 to 15 lag VP2. Sera were collected at 9 to 10 weeks post-inoculation. AHSV-3-neutralizing antibodies were detected in three of the four experimental animals, albeit at low titres of 1:6 to 1:24 (expressed as the serum dilution causing a 50 % plaque reduction). One of the guinea-pigs yielded negative results. These results confirm the findings of Burrage et al. (1993) that AHSV VP2 carries the neutralization-specific epitopes and support the potential of VP2 as a possible subunit vaccine to AHSV. The authors would like to acknowledge the support and assistance of Drs A. A. Van Dijk and B. J. Erasmus and the staff of the Onderstepoort Veterinary Institute as well as S. Cormack for technical support. We also thank Dr D . H . L . Bishop for providing the baculovirus vectors and the Foundation for Research Development and the Agricultural Research Council for financial support. F.T. Vreede was the recipient of a study bursary from the South African Medical Research Council.
References BREMER, C. W. (1976). A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus. Onderstepoort Journal of Veterinary Research 43, 193-200. BREMER, C. W., HUISMANS, H. & VAN DIJK, A. A. (1990). Characterization and cloning of the African horsesickness virus genome. Journal of General Virology 71, 793 799.
BURRAGE, T.G., TREVEJO, R., STONE-MARSCHAT, M. & LAEGREID, W. W. (1993). Neutralizing epitopes of African horsesickness virus serotype 4 are located on VP2. Virology 196, 79%803. CASHDOLLAR, L.W., CHMELO, R., ESPARZA, J., HUDSON, G.R. & JOKLIK, W. K. (1984). Molecular cloning of the complete genome of reovirus serotype 3. Virology 133, 191 196. FUKUSHO, A., RITTER, G.D. & RoY, P. (1987). Variation in the bluetongue virus neutralization protein VP2. Journal of General Virology 68, 2967 2973. GHIASI, H., FUKUSHO, A., ESHITA, Y. & ROY, P. (1987). Identification and characterization of conserved and variable regions in the neutralizing VP2 gene of bluetongue virus. Virology 160, 100-109. GRUBMAN, M. J. & LEWIS, S. A. (1992). Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments. Virology 186, 444451. HOLMES, I.H. (1991). Family Reoviridae. In Classification and Nomenclature of Viruses. Archives of Virology, Supplementum 2, 188-189. HOWELL, P.G. (1962). The isolation and identification of further antigenic types of African horsesickness virus. Onderstepoort Journal of Veterinary Research 29, 139 149. HUISMANS, H. & ERASMUS,B. J. (1981). Identification of the serotypespecific and group-specific antigens of bluetongue virus. Onderstepoort Journal of Veterinary Research 48, 51-58. HUISMANS, H., VAN DER WALT, N. T., CLOETE, M. & ERASMUS, B.J. (1987). Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 157, 172-179. IYUMARU, S. & ROY, P. (1987). Production and characterization of the neutralizing antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector. Virology 157, 472~,79. IWATA, J., YAMAGAWA,M. & ROY, P. (1992). Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. Virology 191, 251-261. KOZAK, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15, 8125-8148. KVXE, J. & DOOL1TTLE, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-120. LAWADA, M.D., ARRAS, M. & SA,NCHEz-VIzcAiNO, J.M. (1993). Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting. Journal of General Virology 74, 81-87. McL~-rOSH, B. M. (1958). Immunological types of horsesickness virus and their significance in immunization. Onderstepoort Journal of Veterinary Research 27, 465-538. M1ZUKOSH1, N., SAKAMOTO, K., IV,rATA,A., TSUCHIYA, T., UEDA, S., APIWATNAKORN, B., KAMADA, M. & FUKUSHO, A. (1993). The complete nucleotide sequence of African horsesickness virus serotype 4 (vaccine strain) segment 4, which encodes the minor core protein VP4. Virus Research 28, 299 306. NEL, L. H. & HUISMANS, H. (1990). A comparison of different cloned genome segments of epizootic haemorrhagic disease virus as serogroup-specific probes. Archives of Virology 110, 103-112. PAERAa'AKUL,U., DE SXASIO,P. R. & TAYLOR, M. W. (1988). A fast and sensitive method for detecting specific viral RNA in mammalian cells. Journal of Virology 62, 113~1135. POSSEE, R. D. & HOWARD, S. C. (1987). Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 15, 10233 10248. RoY, P. (1989). Bluetongue virus genetics and genome structure. Virus Research 13, 17%206. RoY, P., URAKAWA, T., VAN DIJK, A.A. & ERASMUS, B.J. (1990). Recombinant virus vaccine for bluetongue disease in sheep. Journal of Virology 64, 1998 2003. VAN DUK, A. A. (1993). Development of recombinant vaccines against bluetongue. Biotechnology Advances 11, 1-12. VLAK, J.M., KE1NKENBERG, F.A., ZAAE, K. J. M., USMANY, M., KLINGE-ROODE, E. C., GEERVLIET,J. B. F., ROOSIEN,J. & VAN LENT, J. W. M. (1988). Functional studies on the pl0 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing
Short communication a p 10-beta-galactosidase fusion gene. Journal of General Virology 69, 765-776. WE':YER, U. & PoSSEE, R.D. (1991). A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p l0 promoters: coexpression of two influenza virus genes in insect ceils. Journal of General Virology 72, 29672974.
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YAMAGUCHLS., FUKUSHO, A. & ROY~P. (1988), Complete sequence of neutralization protein VP2 of the recent US isolate bluetongue virus serotype 2 : its relationship with VP2 species of other US serotypes. Virus Research 11, 49-58.
(Received 7 April 1994; Accepted 29 July 1994)
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Cloning, characterization and expression of the gene that encodes the major neutralization-specific antigen of African horsesickness virus serotype 3 F. T. V r e e d e t and H . H u i s m a n s * Department of Genetics, University of Pretoria, Pretoria 0001, South Africa
The gene encoding the outer capsid protein, VP2, of African horsesickness virus serotype 3 (AHSV-3) has been sequenced in its entirety from cDNA clones of the segment 2 RNA, and compared with the previously published VP2 gene sequence of AHSV-4. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50-5 % identity to the amino acid sequence of AHSV-4 VP2. Two areas of high variability (approximately 35 % identity) were identified. The N-proximal variable region (amino acids 128 to 309) exhibited significant hydro-
philicity, suggesting a possible role in the determination of the serotype-specific immune response. VP2 of AHSV-3 has furthermore been expressed in a baculovirus expression system. The expressed protein was shown to react specifically with an anti-AHSV-3 serum in Western blots. Antibodies raised in rabbits and guinea-pigs against the recombinant VP2 neutralized the virus in a plaque reduction assay, confirming the identity of VP2 as the major neutralization-specific antigen of AHSV.
African horsesickness (AHS) is a severe, often fatal, viral disease of Equidae endemic to sub-Saharan Africa. The aetiological agent, African horsesickness virus (AHSV), is transmitted by arthropods of the genus Culicoides. AHSV is classified together with a number of other double-stranded RNA viruses, such as bluetongue virus (BTV), in the genus Orbivirus of the family Reoviridae (Holmes, 1991). Nine different AHSV serotypes have been identified with little, if any, cross-neutralization between them (McIntosh, 1958; Howell, 1962). Like BTV, the AHSV particle consists of a doublelayered capsid containing the viral genome, which is composed of 10 dsRNA segments encoding seven structural (VP1 to VP7) and at least three non-structural proteins (Grubman & Lewis, 1992). The outer capsid layer is composed of VP2 and VP5, and surrounds an icosahedral core particle which is composed primarily of VP3 and VP7, with lesser amounts of VP 1, VP4 and VP6 (Bremer, 1976; Bremer et al., 1990; Laviada et al., 1993). Studies with BTV have shown that VP2 is highly variable and is the main determinant of serotype specificity and the neutralization-specific immune response (Huismans
& Erasmus, 1981). VP2 recovered from purified BTV (Huismans et al., 1987) or derived from baculovirus expression vectors (Roy et al., 1990) has been demonstrated to induce neutralizing antibodies and to elicit protection in sheep against virulent viral challenge. VP5 was further found to enhance the neutralization (and protective) immune response (Roy et al., 1990), and recently considerable effort has been exerted in the evaluation of virus-like and core-like particles as subunit vaccines against BTV (Van Dijk, 1993). It has recently been demonstrated that neutralizing epitopes for AHSV are located on VP2 and that antibodies to these epitopes are protective in a neonatal mouse model (Burrage et al., 1993). This suggests that recombinant VP2 bearing these neutralizing epitopes may have efficacy as a subunit vaccine against AHSV. In vitro translation studies have shown that AHSV VP2 is encoded by genome segment 2 (Grubman & Lewis, 1992). The entire sequence of this segment of AHSV-4 has been determined and the deduced VP2 amino acid sequence compared with the cognate proteins of epizootic haemorrhagic disease virus (EHDV) and BTV (Iwata et al., 1992). In this paper, we describe the cloning and sequencing of the VP2 gene of AHSV-3, presenting the first opportunity to qualitatively compare the outer capsid protein gene of two AHSV serotypes and enabling the identification of regions of possible antigenic importance.
t Presentaddress: BiochemistryDivision,OnderstepoortVeterinary Institute, Onderstepoort 0110, South Africa. The sequencedata reported in this paper have been registeredwith the GenBank Data Libraryunder the accessionnumber U01832. 0001-2550 © 1994SGM
3630
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D~LI~dNKLEDGvKCYAYCL~LALYDFHGRDvDGF~GTRTAAIvET~AB~FPDFRSE~S~KFGIDLA~SEESDELFvKKTM~SFSD~GEU~YKF~FG~KTDFKvET~Y~I~SDEV ENLLAC~NSLvGVLRCYUYCLALAIYDFYEGT~DGFKKG~NA~IIET~A~FP~FRREL~EKFGIDLRUKEITRELFVGKSU~SKFUEEGGYGYKFAYGw~RDGFAvUEDYGEILTEKV HRLY~AILDGKE~A~KE~D~PEKYFVD~LYNRCPE$IY~RN~VDPNNK[~[KKRGL~GE~RIFLRDLSHIGMNFKKLLLRL~SK~-~-LHA~GEHIQYHEID~EDFKPCAIAELGLH~T EDLYKG~LLGRK~EDE~DDPESYFY~LYTNEPHR~FL~GKD~DNNITLRS---I~ETTYLSK-RF~SYWYRI~(}~EvTKA~NE~LDUNEKQKPYFEFEYD~FKPC~IGELGIH~T ** , , , ****** ** **** , , • , , , , , * * ***** • e** • •, Y~Y~DLLvGANRGEY~KDAKELVWF~IANTNFNITRPF~RC~:~CAEAEL~LRFHL~TKIFTRY--RGERTSFvDIINEL~EH~YVKHNFPSYKHYYLSv~T~FE~BAIDPL~Fc~A~ YIY~NLL~GRNRGEE~LDSKELVV~D~ASLLNFGAVRSHDRC~I~S~AIEVNLRHALIvB~FSRFDMU~ERETFST~LEKVUE~VKKLRFFPTYRHYYLETL~R~FNDERRLEvDDFYUB ISRNETRESTLKGF~UFTAIvKSERLIDTLFLNFLLwIVFEUENvD~ANKRHPLLIsHEKGLRLIGvDLFNGALSISTGG~IPYLERIc~EEKAQRRLNADELKIKSVVFLTYYUNLSL LYDVQTREQALNTFTDFHRCVESELLLPTLKLNFLL~IVFE~EN~EvNAAYKRHPLLISTAKGLR~IG~D~FN~LSI~d~WIPY~ERU~AESK~QTKLTADELKLKR~FISYYTTLKL ERBAEPRU~FKFEGLTTwIGSNCGGvRDYW~ALPURKPKPGLL~IIYGDDG~A~N~wA~4KNFTAvDC~LGFIYI~RHKLVNKSDFRv~EUKIYNRGRLDRLILI~SGHYTFGNKFLU~ DRRAEPRMSFKFEGLSTw•GSNCGG•R•Y•IQML•TRKPKPGALMVVYAR•SR•E••EAELS•w•••MEGSLGLILVHDSGIINKS•LRARTLKIYNRGSMDTL•LISSGVY•rFGNKFLL• KLLAKTE uaagcggagugacucccgcuccaugugaaucaacuuac KLLAKTE u-agcaacgugacuguugcuccaugugaauacacauac
Fig. 1. Best-fit alignments of the segment 2 non-coding nucleotide sequences (lower case) and the VP2 amino acid sequences (upper case) of AHSV-3 (top) and AHSV-4 (bottom). Conserved residues are indicated by asterisks (identical) or dots (similar).
In addition, we have expressed VP2 of AHSV-3 in a baculovirus expression system and present the results of initial studies of the ability of recombinant AHSV-3 VP2 to induce a neutralization-specific immune response. The strategy employed for the preparation and cloning of cDNA from AHSV-3 segment 2 dsRNA was a modification of the method of Cashdollar et al. (1984) as described by Bremer et al. (1990). The clones were analysed by PstI digestion and hybridization to a Northern blot of AHSV-3 dsRNA. Four overlapping partial cDNA clones (1200, 2000, 1500 and 800 bp), representing the entire AHSV-3 segment 2, were obtained and sequenced. The non-coding nucleotide sequences along with the deduced amino acid sequence of the coding region are shown in Fig. 1 in a direct comparison with VP2 of AHSV-4. Genome segment 2 of AHSV-3 was determined to be 3221 bp in length with a calculated base composition of 14-93% C, 26.12% G, 31.32% A and 27.63 % U. The 5'- and T-terminal hexanucleotides of the AHSV-3 genome segment 2 were 5' GUUUAA and ACUUAC 3' respectively, which supports the consensus AHSV terminal sequences recently proposed by Mizukoshi et al. (1993) that are 5' GUUuA~,A a and cACAUACt,3', based on previous studies involving sequence analyses of the viral RNA species of AHSV. The longest open reading frame (ORF) identified was defined by an AUG at position 13 to 15 and a TAA at position 3184 to 3186, delineating 5' and 3' non-coding regions of 12 bp and 35 bp respectively. The flanking sequences of the AUG codon, CACCAUGG, are comparable to the consensus
sequence for initiation of translation (GCCGCCACCAUGG) identified by Kozak (1987). The AHSV-3 segment 2 gene was deduced to encode a protein composed of 1057 amino acids with a calculated Mr of 123078K. Comparisons of the VP2 genes of AHSV-3 and AHSV4 revealed a 58"7% conservation of nucleotides in type and position, which translates to a 50.5% identity of amino acids in type and position in a best-fit alignment requiring six gaps involving 11 amino acids. When amino acids of similar character were taken into consideration, the similarity index was 71-3 %. The regions that exhibit the greatest similarities are located near the termini and in the centre of the protein (Fig. 2 a). This was confirmed by Diagon analyses (results not shown). These findings correlate with data from extensive comparisons of VP2 of various BTV serotypes which yielded similar percentages and patterns of amino acid conservation (Fukusho et al., 1987; Ghiasi et al., 1987; Roy, 1989; Yamaguchi et al., 1988). Possible functional and conformational roles for the carboxy-terminal and central conserved regions have been suggested. Approximately one-third of the amino acids of the VP2 proteins of both AHSV-3 and AHSV-4 are charged. The hydropathicity profiles of AHSV-3 and AHSV-4 VP2 (Fig. 2b) demonstrated significant similarity, revealing strong hydrophilic characters with a few regions of hydrophobicity evident particularly toward the C terminus. The variability associated with the distinctively hydrophilic domain between amino acids 128 and 309 suggests that the
Short communication (a)
(a)
Regional amino acid identity 67.2% 33.7%
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Amino acids Fig. 2. (a) Graphic representation of the best-fit alignment of the amino acid sequences of VP2 of AHSV-3 and AHSV-4, indicating the regional percentage identical amino acids and (b) the corresponding hydropathicity profiles (Kyte & Doolittle, 1982) of VP2 of AHSV-3 (i) and AHSV-4 (ii).
determinant(s) of the serotype-specific immune response on VP2 may be located in this region within the overall structurally conserved antigen. In order to study the immunogenicity of VP2 of AHSV-3, an Autographa californica nuclear polyhedrosis virus (AcNPV) recombinant containing the full-length clone of the AHSV-3 VP2 gene under control of the p 10 promoter (Vlak et al., 1988) was constructed. A single 3"2 kb product, representing AHSV-3 segment 2, was obtained by PCR amplification of an alkaline sucrose gradient-purified fraction of large AHSV-3 cDNA with synthetic oligonucleotide segment 2 termini-specific primers as described in Nel & Huismans (1990). The primers were designed to include BglII restriction sites to facilitate cloning of the segment 2 gene into the
Fig. 3. Autoradiograph (a) and Western blot (b) of 10% SDS-PAGEseparated extracts of mock-infected (lanes 1) and AHSV-3-infected (lanes 2) Vero cells, AHSV-3 segment 2 recombinant baculovirus (lanes 3), wild-type AcRP23-lacZ (lanes 4) and mock-infected (lanes 5) S. frugiperda ceils. The putative recombinant baculovirus-expressed VP2 protein is marked with an arrow.
baculovirus dual transfer vector pAcUW3 (Weyer & Possee, 1991). Following verification of the correct orientation of the VP2 gene in the transfer vector relative to the baculovirus pl0 promoter, limited sequencing of the termini confirmed the full-length status of this clone. AcNPV VP2 recombinants were obtained by cotransfection of Sf9 cells with the AHSV-3 segment 2 recombinant transfer vector DNA and linearized AcRP23-1acZ DNA, a lacZ recombinant AcNPV derived by Possee & Howard (1987). Putative recombinant viruses that exhibited a white plaque phenotype following exposure to X-gal, were isolated by two rounds of plaque purification. VP2-specific recombinant baculoviruses were identified by dot hybridization of recombinant viral DNA to an AHSV-3 segment 2-specific probe. Using in situ hybridization analyses (Paeratakul et al., 1988), VP2-specific mRNA was detected between 18 h post-infection (p.i.) and 40 h p.i. (results not shown). In order to determine whether AHSV-3 VP2 was synthesized in cells infected with the recombinant AcNPV, the cells were pulse-labelled with [35S]methionine between 28 h and 31 h p.i. and the labelled proteins were analysed by SDS-PAGE (Fig. 3 a). A protein o f M r approximately 116K that comigrated with VP2 from AHSV-3-infected cells, was shown to be synthesized in recombinant AcNPV-infected cells but was absent in mock- or wildtype AcRP23-1acZ-infected cells. In the latter case, the M r l I6K fl-galactosidase protein was expressed and migrated as a band of approximately 120K in SDSPAGE. The band in lane 3 corresponding in apparent M,. to the fl-galactosidase band in lane 4, was identified as a non-specific protein produced in baculovirus-infected cells. Confirmation of the viral origin of the putative VP2
3632
Short communication
protein was obtained by Western immunoblotting of the SDS-PAGE-separated proteins (Fig. 3b). A specific reaction of VP2 with rabbit anti-AHSV-3 serum was observed whereas no interserotype cross-reactivity with horse anti-AHSV-4 serum was exhibited (results not shown). The band of M r approximately 110K in lane 3 which also reacted with the antiserum may represent a truncated form of VP2. These results indicated that AHSV-3 VP2 was expressed during the recombinant virus infection. The yield of expressed VP2 in recombinant AcNPV-infected cells was estimated to be 0'5 to 1 lag/106 cells. This was significantly less than expected, being at least threefold lower than the yield of baculovirus-expressed BTV VP2 (Inumaru & Roy, 1987). The reason for the low yield of AHSV-3 VP2 is currently under investigation. To assess the ability of baculovirus-expressed VP2 to induce a neutralization-specific immune response, plaque-reduction assays were conducted as described by Huismans & Erasmus (1981) with antibodies raised in rabbits and guinea-pigs to recombinant baculovirusinfected cell lysates. The animals were injected intramuscularly with VP2-containing lysates (approximately 60 lag VP2/ml) suspended in an equal volume of Montanide Incomplete Seppic Adjuvant (ISA50). The guinea-pigs each received an initial inoculation of 15 lag VP2 on day 0 followed from day 28 post-inoculation with weekly boosters of 6 to 9 lag VP2. The rabbits were given an initial dose of 30 lag UP2 followed at days 21 and 54 post-inoculation with a further 12 to 15 lag VP2. Sera were collected at 9 to 10 weeks post-inoculation. AHSV-3-neutralizing antibodies were detected in three of the four experimental animals, albeit at low titres of 1:6 to 1:24 (expressed as the serum dilution causing a 50 % plaque reduction). One of the guinea-pigs yielded negative results. These results confirm the findings of Burrage et al. (1993) that AHSV VP2 carries the neutralization-specific epitopes and support the potential of VP2 as a possible subunit vaccine to AHSV. The authors would like to acknowledge the support and assistance of Drs A. A. Van Dijk and B. J. Erasmus and the staff of the Onderstepoort Veterinary Institute as well as S. Cormack for technical support. We also thank Dr D . H . L . Bishop for providing the baculovirus vectors and the Foundation for Research Development and the Agricultural Research Council for financial support. F.T. Vreede was the recipient of a study bursary from the South African Medical Research Council.
References BREMER, C. W. (1976). A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus. Onderstepoort Journal of Veterinary Research 43, 193-200. BREMER, C. W., HUISMANS, H. & VAN DIJK, A. A. (1990). Characterization and cloning of the African horsesickness virus genome. Journal of General Virology 71, 793 799.
BURRAGE, T.G., TREVEJO, R., STONE-MARSCHAT, M. & LAEGREID, W. W. (1993). Neutralizing epitopes of African horsesickness virus serotype 4 are located on VP2. Virology 196, 79%803. CASHDOLLAR, L.W., CHMELO, R., ESPARZA, J., HUDSON, G.R. & JOKLIK, W. K. (1984). Molecular cloning of the complete genome of reovirus serotype 3. Virology 133, 191 196. FUKUSHO, A., RITTER, G.D. & RoY, P. (1987). Variation in the bluetongue virus neutralization protein VP2. Journal of General Virology 68, 2967 2973. GHIASI, H., FUKUSHO, A., ESHITA, Y. & ROY, P. (1987). Identification and characterization of conserved and variable regions in the neutralizing VP2 gene of bluetongue virus. Virology 160, 100-109. GRUBMAN, M. J. & LEWIS, S. A. (1992). Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments. Virology 186, 444451. HOLMES, I.H. (1991). Family Reoviridae. In Classification and Nomenclature of Viruses. Archives of Virology, Supplementum 2, 188-189. HOWELL, P.G. (1962). The isolation and identification of further antigenic types of African horsesickness virus. Onderstepoort Journal of Veterinary Research 29, 139 149. HUISMANS, H. & ERASMUS,B. J. (1981). Identification of the serotypespecific and group-specific antigens of bluetongue virus. Onderstepoort Journal of Veterinary Research 48, 51-58. HUISMANS, H., VAN DER WALT, N. T., CLOETE, M. & ERASMUS, B.J. (1987). Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. Virology 157, 172-179. IYUMARU, S. & ROY, P. (1987). Production and characterization of the neutralizing antigen VP2 of bluetongue virus serotype 10 using a baculovirus expression vector. Virology 157, 472~,79. IWATA, J., YAMAGAWA,M. & ROY, P. (1992). Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences. Virology 191, 251-261. KOZAK, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15, 8125-8148. KVXE, J. & DOOL1TTLE, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-120. LAWADA, M.D., ARRAS, M. & SA,NCHEz-VIzcAiNO, J.M. (1993). Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting. Journal of General Virology 74, 81-87. McL~-rOSH, B. M. (1958). Immunological types of horsesickness virus and their significance in immunization. Onderstepoort Journal of Veterinary Research 27, 465-538. M1ZUKOSH1, N., SAKAMOTO, K., IV,rATA,A., TSUCHIYA, T., UEDA, S., APIWATNAKORN, B., KAMADA, M. & FUKUSHO, A. (1993). The complete nucleotide sequence of African horsesickness virus serotype 4 (vaccine strain) segment 4, which encodes the minor core protein VP4. Virus Research 28, 299 306. NEL, L. H. & HUISMANS, H. (1990). A comparison of different cloned genome segments of epizootic haemorrhagic disease virus as serogroup-specific probes. Archives of Virology 110, 103-112. PAERAa'AKUL,U., DE SXASIO,P. R. & TAYLOR, M. W. (1988). A fast and sensitive method for detecting specific viral RNA in mammalian cells. Journal of Virology 62, 113~1135. POSSEE, R. D. & HOWARD, S. C. (1987). Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Research 15, 10233 10248. RoY, P. (1989). Bluetongue virus genetics and genome structure. Virus Research 13, 17%206. RoY, P., URAKAWA, T., VAN DIJK, A.A. & ERASMUS, B.J. (1990). Recombinant virus vaccine for bluetongue disease in sheep. Journal of Virology 64, 1998 2003. VAN DUK, A. A. (1993). Development of recombinant vaccines against bluetongue. Biotechnology Advances 11, 1-12. VLAK, J.M., KE1NKENBERG, F.A., ZAAE, K. J. M., USMANY, M., KLINGE-ROODE, E. C., GEERVLIET,J. B. F., ROOSIEN,J. & VAN LENT, J. W. M. (1988). Functional studies on the pl0 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing
Short communication a p 10-beta-galactosidase fusion gene. Journal of General Virology 69, 765-776. WE':YER, U. & PoSSEE, R.D. (1991). A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p l0 promoters: coexpression of two influenza virus genes in insect ceils. Journal of General Virology 72, 29672974.
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YAMAGUCHLS., FUKUSHO, A. & ROY~P. (1988), Complete sequence of neutralization protein VP2 of the recent US isolate bluetongue virus serotype 2 : its relationship with VP2 species of other US serotypes. Virus Research 11, 49-58.
(Received 7 April 1994; Accepted 29 July 1994)
