Bluetongue (BT) is a non-contagious infectious disease caused by BTV, a virus of the genus Orbivirus which infects sheep, goats, cattle, deer and camelids [1]. The BTV virion contains a double-stranded RNA genome consisting of 10 segments, which collectively encode 7 structural proteins and 4 nonstructural proteins [2]. The outer capsid protein VP2 of BTV is responsible for receptor binding and haemagglutination, and is targeted by protective immune responses generated following infection of mammalian hosts. While the VP2 protein is a central target of host immune responses to BTV infection, the VP2 protein has been exploited for BT vaccine development with variable results [3]. The observation that VP2 displays considerable sequence variability across the BTV serotypes has complicated the development of effective BT vaccines. The high degree of variability in VP2 amino acid sequences among different BTV serotypes suggests, however, that the VP2 protein may be an excellent antigen to use for the development of BTV serotype-specific antibody reagents.

The impact of BT disease on the international trade of animal products raises important socioeconomic concerns, and BT has consequently been designated a reportable disease by the World Organization for Animal Health [4]. To date, 26 BTV serotypes have been identified [5]. Since 1979, 7 BTV serotypes (BTV1, 2, 3, 4, 12, 15 and 16) have been isolated in China; each serotype causes obvious clinical signs in infected sheep and can inflict severe economic loss in China [6, 7]. There is no or very weak antibody cross-protection between the different BTV serotypes, resulting in the failure of immunization programs in many countries [8]. Therefore, it is imperative to identify which BTV serotypes are circulating in affected areas before initiating vaccination responses.

The aim of this study was to generate a BTV1-specific antibody using the BTV1 VP2 protein as an immunogen, and to subsequently define the B-cell epitope recognized by the antibody. The information and reagents described in this report will facilitate the development of BTV diagnostic tools and further our understanding of the antigenic structure of VP2 protein which will benefit the rationale design of BTV vaccines.

BTV1 VP2 protein for immunization of mice was produced using a prokaryotic expression system. The full length VP2 coding sequence was amplified using primers designed according to the sequence of the BTV1 SZ97/1 strain (GenBank JN848760.1) and flanked by BamHI and HindIII restriction endonuclease sites. The recombinant plasmid was then transformed into E. coli BL21 competent cells to express recombinant VP2 protein. After induction of recombinant protein production, the bacteria were spun at 10000 g for 10 min at 4 °C, and lysed by sonication in phosphate buffered saline (PBS) to release recombinant VP2 protein. The recombinant VP2 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB) using BTV1-positive mouse sera as the primary antibody with an HRP-conjugated goat anti-mouse secondary antibody. The WB was performed as described previously. Gel-purified recombinant VP2 protein was used as antigen to immunize mice for the generation of BTV1-specific mab according to standard procedures [9].

BTV1 VP2 protein for hybridoma screening was produced using the Bac-to-Bac® Baculovirus Expression System (Invitrogen). Supernatant containing recombinant baculovirus was harvested 4 days following transfection of Sf9 insect cells with recombinant bacmid DNA encoding the BTV1 VP2 protein. The baculovirus was used to express recombinant VP2 protein in Sf9 insect cells. SDS-PAGE followed by WB analysis using BTV1-positive mouse sera as the primary antibody with an HRP-conjugated goat anti-mouse secondary antibody was performed to confirm the production of VP2 protein in the insect cells. As cloning of VP2 into the pFastBacTMHTA expression vector places an N-terminal histidine tag in frame with the VP2 protein, recombinant VP2 protein was purified using Ni-nitrilotriacetic acid affinity chromatography (Qiagen) according to the manufacturer’s instructions.

The hybridoma lines were screened by indirect ELISA against the purified recombinant VP2 protein produced in Sf9 insect cells as described previously [10]. Clones were screened three times by limiting dilution to subclone the positive hybridoma lines. Selected clones were injected into the peritoneal cavities of pristane (Sigma, USA)-primed BALB/c mice to generate ascites fluid. Immunoglobulin subtypes were determined using the Mouse MonoAb-ID Kit (HRP) (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

ELISA-positive clones were further characterized by WB and immunofluorescence assay (IFA). For the WB analysis, lysates of BHK21 cells and BHK21 cells infected with BTV1 were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Hybridoma supernatant was used as a primary antibody source followed by an HRP-conjugated goat anti-mouse IgG secondary antibody. For the IFA, BHK21 cells were grown in a 96-well cell culture microplate. Cells were infected with the indicated BTV (BTV1-24) when BHK21 cells were approximately 90 % confluent. Thirty-six hours later, Hybridoma supernatant was used as a primary antibody and a FITC-conjugated goat anti-mouse IgG was used as a secondary antibody, and the microplate was viewed on a fluorescence microscope.

Ninety-six oligonucleotides corresponding to the coding sequence of a series of peptides from the full-length BTV1 VP2 protein were synthesized and cloned into the EcoRI/SalI sites of the pMAL™-C4x expression vector. In this system, the peptide sequence is expressed as an MBP fusion protein following transformation of E. coli BL21 cells and IPTG induction of protein production. The production of each BTV1 VP2-derived peptide in the series was confirmed by SDS-PAGE. The expressed peptides were 16 amino acid (a.a.) residues long and each peptide overlapped the previous peptide sequence by 6 a.a. residues. The series of recombinantly produced VP2-derived peptides were screened using an indirect ELISA as described above to define the peptide epitope recognized by the mab 4B6.

To evaluate the conservation of the identified epitope among BTV1 isolates, we aligned sequences corresponding to the region encompassing the mab 4B6 peptide epitope of the VP2 protein from the 21 BTV1 isolates for which sequence information was available through NCBI. The alignment was repeated using available sequence information on the different BTV serotypes (BTV1-26). Peptides corresponding to the homologous region of the VP2 protein of BTV25 and BTV26 were synthesized to further evaluate the serotype specificity of the mab 4B6 in an indirect ELISA.

We generated recombinant VP2 protein in E. coli and insect cells to use as an immunogen to generate and screen VP2-specific monoclonal antibodies. The recombinant BTV1 VP2 protein produced in E. coli BL21 was recognized by BTV1-positive mouse sera in WB (Fig. 1a). The predominant band was approximately 156 kDa, which is in accordance with the predicted mass of VP2. Following protein induction in E. coli, the majority of VP2 protein was associated with the insoluble inclusion bodies (Fig. 1a, lane 1), with considerably less VP2 protein detectable in the soluble fraction (Fig. 1a, lane 2). As the majority of VP2 protein was found in the inclusion bodies, recombinant protein could not be purified using affinity chromatography on an amylose resin. As an alternative approach, we purified VP2 protein by separating the bacterial lysate containing VP2 protein on an acrylamide gel, excising the band corresponding to full-length VP2 protein, and eluting VP2 protein from the acrylamide (Fig. 1b, lane 1).

Fig. 1
figure 1

Characterization of recombinant VP2 protein and the monoclonal antibody 4B6. (a) Western blot analysis of recombinant VP2 protein produced in E. coli BL21 and Sf9 insect cells using BTV1-positive mouse sera. M, Molecular weight marker; Lane 1, Pellet of bacterial cell lysate from induced VP2-expressing E. coli BL21 cells; Lane 2, Supernatant from induced VP2-expressing E. coli BL21 cells; Lane 3, Pellet of bacterial cell lysate from uninduced VP2-expressing E. coli BL21 cells; Lane 4, MBP-tagged protein expressed by empty pMALTM-c4x vector; Lane 5, Sf9 insect cells infected with recombinant baculovirus encoding the BTV1 VP2 protein; Lane 6, Sf9 insect cells infected with wild-type baculovirus; Lane 7, uninfected Sf9 insect cells. (b) SDS-PAGE analysis of recombinant VP2 protein after purification. Acrylamide gel purification methods were used to purify recombinant VP2 protein expressed in E. coli BL21 cells. Recombinant VP2 protein expressed in Sf9 was purified by Ni-nitrilotriacetic acid affinity chromatography. M, Molecular weight marker; Lane 1, purified recombinant VP2 protein expressed in E. coli BL21 cells; Lane 2, recombinant VP2 protein expressed in E. coli BL21 before purification; Lane 3, recombinant VP2 protein expressed in Sf9 cells before purification; Lane 4, purified recombinant VP2 protein expressed in Sf9 cells. (c) The mab 4B6 recognizes recombinant VP2 protein. Cell lysates from uninfected BHK21 cells (lane 1) and BHK21 cells infected with BTV1 (lane 2) were evaluated for reactivity with the mab 4B6 using a Western blot. M, Molecular weight marker

The recombinant BTV1 VP2 protein produced in Sf9 insect cells was recognized by BTV1-positive mouse sera by WB (Fig. 1a, lane 5). The target band was approximately 116 kDa by SDS-PAGE analysis, which is in agreement with the predicted mass of VP2. There was a difference in molecular weight of VP2 produced in E. coli and Sf9 insect cells because the mass of MBP tag (45 kDa) and His tag (6 kDa) are different. Following affinity chromatography using Ni-nitrilotriacetic acid, a single band corresponding to the full length VP2 protein was apparent (Fig. 1b, lane 4).

We immunized mice with the recombinant VP2 expressed in E. coli and generated a panel of VP2-specific antibody-secreting hybridomas. The mab designated as 4B6 was selected for characterization based on strong reactivity against recombinant BTV1 VP2 protein in an indirect ELISA (data not shown). The mab 4B6 recognized VP2 protein produced during infection of BHK21 cells with BTV1 by WB (Fig. 1c, lane 2). The size of the protein recognized by mab 4B6 is approximately 110 kDa, corresponding to the predicted mass of the natural VP2 protein. Further characterization by IFA revealed that mab 4B6 bound BHK21 cells infected with BTV1, but did not react with BHK21 cells infected with other BTV serotypes (Fig. 2). The mab 4B6 consisted of an IgG1 heavy chain paired with a κ-type light chain. These results indicate that the IgG1 mab 4B6 is highly-specific for the VP2 protein of BTV1.

Fig. 2
figure 2

mab 4B6 reacts with cells infected with BTV1. Indirect immunofluorescence was performed using cells infected with BTV1-24, or uninfected BHK-21 cells as a negative control. The mab 4B6 supernatant was used as the primary antibody, and FITC-conjugated goat anti-mouse IgG was used as a secondary antibody. The positive control was incubated with sera from BTV1-positive mice sera along with the appropriate FITC-conjugated secondary antibody

We next screened mab 4B6 against a series of 96 overlapping peptides derived from the amino acid sequence of the BTV1 VP2 protein in order to map the reactivity of mab 4B6. Expression of each peptide fusion protein was confirmed by SDS-PAGE (Supplementary Fig. S1). The mab 4B6 reacted with a peptide corresponding to a.a. 111–126 of the BTV1 VP2 protein in an indirect ELISA (data not shown). Therefore, the linear epitope recognized by mab 4B6 is contained within the BTV1 VP2 sequence 111DSMDAQPLKVGLDDQS126. To further resolve the peptide sequence recognized by mab 4B6, we performed additional analysis using a series of progressively truncated peptides (Fig. 3a). This analysis showed that mab 4B6 recognized peptides containing an 8 a.a. core, corresponding to the BTV1 VP2-derived sequence 115AQPLKVGL122 (Fig. 3a). The truncation of additional amino acids at either end of this core sequence abolished mab 4B6 reactivity, demonstrating that the terminal alanine and leucine residues were critical to 4B6 binding. These results demonstrate that mab 4B6 recognizes a linear peptide epitope corresponding to the VP2 sequence 115AQPLKVGL122.

Fig. 3
figure 3

Identification of the minimal peptide epitope recognized by mab 4B6. (a) A peptide encompassing the mab 4B6 epitope and a series of truncated peptides were used to map the minimal peptide epitope required for 4B6 recognition using an indirect ELISA. Peptides screened include: BTV1-16DS, DSMDAQPLKVGLDDQS; BTV1-14SQ, SMDAQPLKVGLDDQ; BTV1-12MD, MDAQPLKVGLDD; BTV1-10DD, DAQPLKVGLD; BTV1-8AL, AQPLKVGL; BTV1-7AG, AQPLKVG; BTV1-7QL, QPLKVGL; BTV25-8II, IQPLKVEI; BTB26-8VI, VQPLKILI. An anti-porcine IFN-γ mab was used as a control antibody. (b) Amino acid alignment of the region encompassing the mab 4B6 epitope for 21 BTV1 isolates. (c) Amino acid alignment from the region encompassing the mab 4B6 epitope of different BTV serotypes (BTV1-26). Alignment was performed using Lasergene analysis software. Black dots indicate the presence of the same residue found in the BTV1 sequence at that position

Alignment of the VP2 amino acid sequences of BTV1 isolates revealed that the peptide epitope recognized by mab 4B6 is highly conserved among BTV1 isolates, including isolates collected in South Africa, Australia, Nigeria, and Cameroon (Fig. 3b). Among the different BTV1 strains, one isolate, AFH36049.1, had two substitutions in the region corresponding to the mab 4B6 epitope at a.a. position 120 (V→I) and a.a. position 121 (G→E). Despite these two a.a. changes, this BTV1 variant epitope consisting of the peptide sequence AQPLK-I-E-L was recognized by mab 4B6 in an indirect ELISA (Fig. 3a), supporting the concept that mab 4B6 specifically recognizes BTV1 serogroup isolates.

In contrast to the highly conserved nature of the 4B6 epitope among BTV1 isolates, this region of the VP2 protein displayed a higher degree of variability among other BTV serotypes (Fig. 3c), suggesting the mab 4B6 recognizes a serotype-specific VP2 epitope present only in BTV1 isolates. To test this, we synthesized peptides corresponding to homologous regions of the BTV25 and BTV26 VP2 protein and tested the peptides for reactivity against the mab 4B6. Whereas mab 4B6 reacted with peptide epitopes derived from BTV1 isolates, the mab 4B6 did not react with corresponding peptides derived from BTV25 and BTV26 VP2 (Fig. 3a). Collectively, these results demonstrate that the peptide epitope recognized by mab 4B6 is a BTV1-specific antigen and that this monoclonal antibody may be used to serologically distinguish BTV1 isolates from other BTV serotypes.

Antibodies that neutralize BTV predominantly recognize the VP2 protein and some VP2 antibody recognition sites that mediate BTV neutralization are serotype-specific [11]. While the mab 4B6 recognizes the VP2 protein, it does not abolish infectivity of BTV1 in a microneutralization assay (data not shown), demonstrating that 115AQPLKVGL122 is not a neutralizing antibody epitope.

BT is a vector-borne viral disease of ruminants that is endemic in tropical and subtropical countries. The epidemic range of BTV is expanding as the climate warms. Identification of BTV serotypes is important to match vaccination programs with circulating BTV serotypes and to study BTV epidemiology. Traditional methods to diagnose and distinguish BTV serotypes, such as virus isolation, serology, virus neutralization tests are slow and often yield inconclusive results [12]. In contrast, serotype-specific antibodies can be used to provide a rapid, sensitive and reliable method for the identification of BTV serotypes. Antibodies specific for select BTV serogroups have been generated [13, 14], but no antibodies specific for BTV1-specific antibodies have been reported. In this study, we report the generation and characterization of a BTV1-specific mab 4B6 and defined the linear peptide epitope. In consideration of that we don’t have many strains in each BTV serotype to verify the reactivity of mab 4B6, we compared the epitope in different BTV serotypes. The results show that the epitope is BTV1-specific and this monoclonal antibody may be applied to the development of diagnostic tools for BTV infection in China.