Bluetongue (BT) is an infectious, non-contagious viral disease leading to substantial economic losses to the livestock industry. Bluetongue virus (BTV) is an economically important arbovirus that affects domestic and wild ruminants and various other Artiodactyla. BTV (F: Reoviridae, G: Orbivirus) is a complex, non-enveloped virus with 7 structural and 4 nonstructural proteins encoded by 10 segments of dsRNA and has 24 serotypes with the involvement of Culicoides vectors in its transmission. All the serotypes share a common VP7 protein, but vary in their VP2 and VP5 proteins which produce neutralizing antibody in the infected animals. BTV exhibits a distinct evolutionary lineage or topotype making the diagnosis more difficult.
In field conditions, sheep is the primary host and the tentative diagnosis of BT relies on the disease epizootiology, vector distribution, clinical symptoms and pathological lesions. Clinical symptoms are not sufficient to determine quarantine and implementation of a control program. Isolation of the virus in embryonated chicken eggs or cell cultures is also required, along with identification of the viral antigens in the tissues/body fluids or antibodies in sera by various diagnostic assays such as immunofluorescence, immunoperoxidase staining , agar gel immuno-diffusion test (AGID), serum neutralisation test (SNT), fluorescent antibody test (FAT), or enzyme linked immunosorbent assay (ELISA)[1, 10, 15, 16]. Additionally, polymerase chain reaction (PCR) and quantitative PCR (QPCR) for detection of viral nucleic acid as well as serogrouping, serotyping and topotyping of BTV isolates have been reported.
Bluetongue cELISA is one of the OIE prescribed tests for international trade of ruminants, while, bluetongue AGID test is an alternative test . BT is enzootic in India and its occurrence is sporadic. The presence of 21 serotypes in the country has complicated the diagnosis, serotyping and characterization of the virus as well as implementation of a control strategy. Currently, the sero-surveillance and sero-diagnosis of BT is being carried out using imported ELISA kits, which are expensive and often associated with the risk of importing exotic pathogens. In view of the above, the present study was undertaken to produce and characterize monoclonal antibodies against BTV23.
Each of the six BTV serotypes BTV1, 2, 15, 17, 18 and 23 produced specific cytopathic effects (CPE) after 36 h post infection (PI) which was completed after 72 h PI. Furthermore, BTV1, 2, 15, 17, 18 and 23 were also confirmed by polyacrylamide gel electropho-resis (Data not shown). BTV23 was produced in bulk in the BHK21 cell line for further study. The titre of the virus was 106.5TCID50/mL. The protein content of the purified virus was 1.75 mg/mL and the purity of the virus was 90%. The immunized mice were test bled before fusion and tested for the antibodies against BTV in iELISA (Fig. 1).
The reactivity of hybridoma culture supernatants with purified BTV23 and BHK21 in iELISAis shown in figure 2A. Similarly, all the BTV reactive clones (n = 24) were further tested for their reactivity with rVP7. Likewise, all the selected primary hybridoma clones were checked for their specificity to rVP7 protein in indirect ELISA. Out of 24 clones, only eight clones (4A7, 4A10, 4A11, 4C8, 4G9, 4H11, 5B5 and 5D5) were found to specifically react with rVP7 protein (Fig. 2B).
Of the 24 MAbs isotypes, the majority of them were found to be IgG class belonging to different subclasses. The remainder, 3G7, 4A7, 5D5, 4E5, 4G6, 4G9 belonged to the IgM class. The details of isotypes, reactivity with BTV23, rVP7, titres and their diagnostic applications are shown in Table 1.
Table 1. Characteristics of bluetongue virus specific monoclonal antibodies
The titres and titration curves of some of the MAbs are presented in Table Ⅰ and Figure 3. Overall, the MAbs with sigmoid curves (4A7, 5D5 with BTV23 antigen and 4A10, 4A11 and 4H11 with purified rVP7 protein of BTV23) had high antibody titres followed by MAbs with linear curves (4G9 and 5B5 with BTV23 antigen). Low antibody titres were observed in MAbs having concave reactivity curves (4A10, 4A11, 4C8 and 4H11 with BTV23 antigen; 4A7, 4C8, 4G9 and 5B5 with rVP7 protein of BTV23) which reacted only at lower dilution or undiluted form or rarely when diluted 1:2 (Fig. 3).
Of the 8 clones tested for their suitability in c‑ELISA, only the clone designated as 4A10 was found to be possibly suitable as inhibition of the rest of the clones was much less than the 4A10 clone with BTV23 hyperimmune serum. All the 24 serotype specific sera of BTV inhibited the MAb with variable percentage inhibition ranging from 16.6% with BTV 12 serotype to 78.9% with BTV16 serotype at the lowest dilution of serum (which indicates the specificity of the MAb). A highest PI of 78.9% was observed with HIS of BTV16 followed by 76.2% with BTV5 serotype and the least was observed with BTV12 (16.6%), at lowest dilution of serum. The control or healthy sera also showed certain level of inhibition at low dilution. The PI was decreased as the dilution of the sera was increased (Fig. 4). Similarly, variable levels of PI were observed with a few of the selected field sera of sheep (Table 2). Epizootic haemorrhagic disease, Ibaraki and Akabane viruses have not yet reported in the country, so the cross reactivity with these viruses has not been ascertained.
Figure 4. Inhibition of MAb by individual BTV serotype specific serum in c‑ELISA. A: BTV1-6 and healthy sheep serum; B: BTV7-12; C: BTV13-18; D: BTV19-24.
Table 2. Field and representative hyperimmune sera showing variable percentage of inhibition in cELISA using whole BTV virus and rVP7 protein