Mapping of B-cell epitopes of hemagglutinin protein of rinderpest virus

Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.     

Cytotoxic T cell epitope in cattle from the attachment glycoproteins of rinderpest and peste des petits ruminants viruses

The surface glycoproteins of rinderpest virus (RPV) confer protective immunity in cattle. We demonstrated that cattle immunized with a recombinant extracellular baculovirus expressing the hemagglutinin (H) protein of RPV (rECV-H) generate virus neutralizing antibody responses, bovine leukocyte antigen (BoLA) class II restricted helper T cell responses and BoLA class I restricted cytotoxic T cell (CTL) responses against RPV-H and hemagglutinin-neuraminidase (HN) glycoprotein of closely related Peste des petits ruminants virus (PPRV). In this study, employing autologous skin fibroblasts transiently expressing truncations of H and HN in a BoLA class I restricted lymphoproliferation assay, we have mapped a highly homologous domain (amino acids 400-423) on these proteins harboring a CTL epitope. Subsequently, based on sequence comparison with available BoLA class I binding motifs, we have identified a BoLA-A11 binding motif (amino acids 408-416) in the stimulatory domain. Autologous cells pulsed with a synthetic peptide corresponding to this sequence stimulated CTLs from rECV-H immunized as well as tissue culture attenuated RPV vaccinated cattle of different breeds and parentage. This is the first epitope identified in cattle on the attachment glycoproteins of RPV and PPRV.     

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.     

The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.

The T-helper response to the measles virus nucleoprotein (NP) has been studied in mice. The T-cell proliferative response was measured in lymphocytes from mice immunized with either a vaccinia measles-NP recombinant virus or a mouse neuro-adapted measles virus. A T-cell response was obtained with lymphocytes from H2d or H2k mice when stimulated with either measles virus or the NP expressed in bacteria. The response was CD4+ specific. The T-helper epitopes were mapped using truncated NP peptides. The major epitopes in both H2d and H2k mice were determined to be between amino acids 67-98. A further T-cell epitope (between amino acids 457-525) was identified when H2d mice were immunized with measles virus. Studies to quantitate the precursor cells for these epitopes confirmed that the region 67-98 of NP was immunodominant in both haplotypes immunized with the vaccinia-NP recombinant virus, whereas an additional major epitope was observed in the measles virus-infected H2d mice. The primary structure of the epitopes determined here are compared to predicted T-cell epitope motifs.     

Recombinant hemagglutinin protein of rinderpest virus expressed in insect cells induces cytotoxic T-cell responses in cattle.

Rinderpest virus (RPV), a member of the genus Morbillivirus within the Paramyxoviridae family, causes a highly contagious and often fatal disease known as rinderpest in wild and domestic ruminants. The envelope of the virus contains two surface glycoproteins, namely the hemagglutinin (H) and the fusion (F) proteins, both of which have been shown to confer protective immunity in animals. In this paper, we demonstrate that single administration of low doses of recombinant H protein of RPV expressed in insect cells in the form of extracellular virus induces long lasting bovine leukocyte antigen class I restricted cytotoxic T-cell (CTL) responses in cattle in the absence of adjuvant. This is the first report of CTL responses in cattle against one of the protective antigens of RPV.     

Immunodominant epitopes in Babesia bovis rhoptry-associated protein 1 that elicit memory CD4(+)-T-lymphocyte responses in B. bovis-immune individuals are located in the amino-terminal domain

Babesia bovis rhoptry-associated protein 1 (RAP-1), which confers partial protection against B. bovis challenge, is recognized by antibodies and T lymphocytes from cattle that have recovered from infection and are immune to subsequent challenge. RAP-1 is a 60-kDa protein with an N-terminal (NT) region that contains four cysteine residues conserved among all Babesia RAP-1 family members and a C-terminal (CT) region that contains multiple, degenerate, tandem 23-amino-acid (aa) repeats. To define the location of CD4(+)-T-cell epitopes for vaccine development using a recombinant protein or minigene construct, a series of truncated recombinant RAP-1 proteins and peptides were tested for stimulation of T-cell lines derived from B. bovis-immune cattle. CD4(+)-T-cell lines from three B. bovis-immune cattle with different DRB3 haplotypes responded to the NT region of RAP-1, whereas T cells from only one animal responded weakly to the CT region. T-cell lines from the three individuals recognized two to six NT-region peptides spanning aa 134 to 316 and representing at least four dominant epitopes. Using RAP-1-specific CD4(+)-T-cell clones, two NT-region epitopes, EYLVNKVLYMATMNYKT (aa 187 to 203) and EAPWYKRWIKKFR (aa 295 to 307), and one CT-region repeat epitope, FREAPQATKHFL, which is present twice at aa positions 391 to 402 and 414 to 425, were identified. Several peptides representing degenerate repeats of the agonist CT-region peptide FREAPQATKHFL neither stimulated responses of T-cell clones specific for this peptide nor inhibited responses to the agonist peptide. Upon stimulation with specific antigen, T-cell clones specific for NT or CT epitopes produced gamma interferon. The presence of T-helper-cell epitopes in the NT domain of RAP-1, which is highly conserved among otherwise antigenically different strains of B. bovis, supports the inclusion of this region in vaccine constructs to be tested in cattle.     

The nucleotide and deduced amino acid sequence of the M gene of phocid distemper virus (PDV). The most conserved protein of morbilliviruses shows a uniquely close relationship between PDV and canine distemper virus.

The nucleotide sequence of the matrix gene (M) of a recently identified morbillivirus, phocid distemper virus (PDV), was determined and the amino acid composition deduced. The M gene of PDV shared many characteristics with the corresponding gene in other morbilliviruses. The nucleotide homology with the closely related canine distemper virus (CDV) was maximum at 67% followed by measles virus (MV) (58%) and rinderpest virus (RPV) (56%). The length of the 5' long untranslated region of PDV (408) was similar to that of CDV (406) but was somewhat shorter than that of MV (425) and RPV (437). The deduced matrix protein of PDV showed structural characteristics similar to the corresponding proteins of other morbilliviruses. PDV and CDV M proteins showed a remarkably high amino acid homology of 90%. The percent amino acid homology among other morbilliviruses was between 73-77%. The M protein was the most highly conserved protein among all morbilliviruses viral components.     

Structural and functional studies of the measles virus hemagglutinin: identification of a novel site required for CD46 interaction

The entry of measles virus (MV) into human cells is mediated by the initial attachment of the viral hemagglutinin (HA) to the complement regulatory protein CD46. Two subdomains, one each within CD46 short consensus repeats (SCRs) 1 and 2, are responsible for this interaction. However, little is known about the regions within MV HA needed for a high-affinity CD46 interaction. To better define the HA-CD46 interaction, we took three approaches: chimeric domain swapping, peptide scanning, and alanine scanning mutagenesis. Chimeras of MV HA and the closely related rinderpest virus (RPV) HA were generated and tested for cell surface expression and the ability to hemadsorb CD46+ red blood cells (RBC). Exchanges with the N terminus of RPV were tolerated as MV HA could be replaced with RPV HA up to amino-acid position 154. However, both larger swaps with RPV and a small RPV HA replacement at the C terminus aborted cell-surface expression. Peptide scanning with 51 overlapping peptides derived from three MV HA regions showed one peptide, corresponding to MV HA amino acids 468-487, blocked hemagglutination of African green monkey (AGM) RBCs and inhibited MV infection of Chinese hamster ovary cells (CHO) expressing human CD46. Alanine scanning mutants mapped sites on the MV HA that were not required for trafficking to the cell surface or function in hemagglutination as well as a novel site required for CD46 interaction, amino acids 473-477.     

Characterization of immunodominant linear B-cell epitopes on the carboxy terminus of the rinderpest virus nucleocapsid protein

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N(1-525)), an amino-terminal construct (N(1-179)), and a carboxy-terminal construct (N(414-496)), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N(414-496) was much more antigenic than GST-N(1-179) when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues (440)VPQVRKETRASSR(452) (site 1), (479)PEADTDPL(486) (site 2), and (520)DKDLL(524) (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.     

Molecular Cloning and Sequence Analysis of the Phosphoprotein (P) Gene of the Lapinized Rinderpest Virus

We determined the nucleotide sequence of the coding region for the phosphoprotein (P) gene of the L strain of rinderpest virus (RPV). The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids, while use of the second ATG would produce a C polypeptide of 177 amino acids. In addition, the insertion of an extra G residue at the editing site generates an alternative mRNA potentially encoding the V protein of RPV. Homology comparisons of the P, C and V proteins among various viruses suggest that RPV is closer to measles virus (MV) than to canine distemper virus (CDV). Alignment of the sequences unique to the V protein revealed that the cysteine residues are well conserved among RPV, MV and CDV, and form a zinc finger-like motif.     

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.     

Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.     

Antigenic and immunogenic investigation of B-cell epitopes in the nucleocapsid protein of peste des petits ruminants virus

Attempts were made to identify and map epitopes on the nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) (Nigeria75/1 strain) using seven monoclonal antibodies (MAbs) and deletion mutants. At least four antigenic domains (A-I, A-II, C-I, and C-II) were identified using the MAbs. Domains A-I (MAb 33-4) and A-II (MAbs 38-4, P-3H12, and P-13A9) were determined to be located on the amino-terminal half (amino acids [aa] 1 to 262), and domains C-I (P-14C6) and C-II (P-9H10 and P-11A6) were within the carboxy-terminal region (aa 448 to 521). Nonreciprocal competition between A-II MAbs and MAbs to C-I and C-II domains was observed, indicating that they may be exposed on the surface of the N protein and spatially overlap each other. Blocking or competitive enzyme-linked immunosorbent assay studies using PPRV serum antibodies revealed that epitopes on the domains A-II and C-II were immunodominant, whereas those on the domains A-I and C-I were not. The competition between MAb and rinderpest virus (RPV) serum antibodies raised against RPV strain LATC was found in two epitopes (P-3H12 and P-13A9) on the domain A-II, indicating that these epitopes may cause cross-reactivity between PPRV and RPV. Identification of immunodominant but PPRV-specific epitopes and domains will provide the foundation in designing an N-protein-based diagnostic immunoassay for PPRV.     

Analysis of the Neutralizing Antibody Response to the Measles Virus Using Synthetic Peptides of the Haemagglutinin Protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310–325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35–58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

Identification and characterization of the neutralization epitope(s) of the hepatitis E virus

The neutralization epitope(s) of the hepatitis E virus (HEV) was studied by an in vitro neutralization assay using antibodies obtained by immunization of mice with 51 overlapping 30-mer synthetic peptides spanning the region 221-660 amino acids (aa) of the HEV open reading frame 2 encoded protein (pORF2) and 31 overlapping recombinant proteins of different sizes derived from the entire pORF2 of the HEV Burma strain. Antibodies against synthetic peptides and short recombinant proteins of approximately 100 aa did not neutralize HEV, suggesting the HEV neutralization epitope(s) is conformation-dependent. However, one recombinant protein of approximately 400 aa in length comprising the pORF2 sequence at position 274-660 aa as well as all truncated derivatives of this protein containing region 452-617 aa elicited antibodies, demonstrating HEV neutralizing activity. These findings establish for the first time that the minimal size fragment, designated pB166, that can efficiently model the neutralization epitope(s) is 166 aa in length and is located at position 452-617 aa of the HEV pORF2. Additionally, antibodies against pB166 were found to cross-neutralize three different HEV genotypes, suggesting that a common neutralization epitope(s) may exist within the different HEV genotypes. Thus, recombinant proteins constructed in this study may be considered as potential candidates for the development of an HEV subunit vaccine as well as for the development of highly sensitive and specific diagnostic tests.     

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I-III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.     

Characterization of the complete genomic sequence of the rinderpest virus Fusan strain cattle type, which is the most classical isolate in Asia and comparison with its lapinized strain

In this study, we characterized the rinderpest virus (RPV) Fusan strain cattle type (B), which is the most classical isolate in Asia, by complete genomic sequence analysis and compared it with its lapinized Nakamura III (L) strain. The transversion rates of the M, F, and H genes were higher than those of other genes. In contrast, the deduced amino acid (aa) substitution rates of the P, C, and V genes were higher than those of other genes, although their transversion rates were not higher. The characteristic nucleotide (nt) or aa residues of the cattle-virulent B and Kabete 'O' strains were observed in the P/C/V, M, and L genes. According to these results, we speculate that nt/aa substitution in the P/C/V genes is one of the key determinants for the difference in the pathogenicity to cattle of the B and L strains.     

Cloning and sequence analysis of the hemagglutinin gene of the virulent strain of rinderpest virus

We have cloned the cDNA of the hemagglutinin (HA) gene of the virulent (Kabete O) strain of rinderpest virus and produced a comparative analysis of its sequence with that of the HA genes of rinderpest (lapinized strain) and measles viruses. The gene has an open reading frame of 1827 nucleotides, and the derived protein is 609 amino acids long with a calculated molecular weight of 68,006. The Kabete O HA polypeptide is identical in length to the HA of the lapinized strain of rinderpest virus and has a similar hydropathy profile. The nucleotide divergence between the lapinized and the Kabete O HA genes is 11.4% within the coding region, and 34.3% in the 3' untranslated region. The two rinderpest HA polypeptides differ at 74 amino acid residues for a divergence of 12.2%. Three-way comparison of the two rinderpest HA molecules with the measles virus HA polypeptide indicates that 56.6% of the residues are conserved.     

Competitive enzyme-linked immunosorbent assay based on monoclonal antibody and recombinant hemagglutinin for serosurveillance of rinderpest virus

A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects antibodies unique to rinderpest virus (RPV) has been developed. This test can differentiate antibodies against RPV and those against peste des petits ruminants virus. The recombinant RPV hemagglutinin (H)-protein C-ELISA (recH C-ELISA) is based on the ability of a well-characterized monoclonal antibody (MAb) produced with the soluble, secreted form of the H protein (Sec H protein) of RPV made in a baculovirus expression system to compete with the binding of RPV antibodies in the serum of vaccinated or infected, recovered animals to the Sec H protein. The B-cell epitope recognized by the MAb corresponds to amino acids 575 to 583 on the H protein, which is not present on the antigenically closely related peste des petits ruminants virus hemagglutinin-neuraminidase protein. Initially, a positive-negative threshold cutoff value for percent inhibition of 34 was established with 500 known RPV-negative serum samples. The recH C-ELISA was developed with the enzyme immunoassay software of a commercial RPV C-ELISA kit. Comparative analysis of the test results for 700 serum samples obtained with the commercial kit gave a sensitivity of 112.4% and a specificity of 72.4%. Variations in percent inhibition values were observed for the two assay systems. These variations may have been due to the undefined amount of antigen present in the commercial kit as well as the use of a different MAb. The recH C-ELISA detected more positive serum samples compared to the number detected by the commercial kit, with the results confirmed by a virus neutralization test. Thus, recH C-ELISA is a sensitive tool for RPV serosurveillance in disease eradication programs.     

Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N_1-525), an amino-terminal construct (N_1-179), and a carboxy-terminal construct (N_414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N_414-496 was much more antigenic than GST-N_1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues ⁴⁴⁰VPQVRKETRASSR⁴⁵² (site 1), ⁴⁷⁹PEADTDPL⁴⁸⁶ (site 2), and ⁵²⁰DKDLL⁵²⁴ (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.