Polyclonal Antibody Production for Ovine Lentivirus Detection

Pairwise protein sequence alignments for the identification of conserved regions in the envelope (Env) proteins of exJSRV, the MVV and CAEV (sequences retrieved from the National Centre for Biotechnology Information, NCBI) were conducted using the Geneious prime software version 2020 (https://www.geneious.com, accessed on 6 December 2021). The region of interest to produce polyclonal antibodies against exJSRV was chosen according to Liu et al. [37 (link)]. The prediction of antigenic epitopes was conducted using the Antibody Epitope Prediction tools of the Immune Epitope Database and Analysis Resource (http://www.iedb.org/home_v3.php) and the Antigen Profiler tool from ThermoFisher Scientific (https://www.thermofisher.com/ch/en/home/life-science/antibodies/custom-antibodies/custom-antibody-production/antigen-profiler-antigen-preparation.html, accessed on 20 January 2021) (Supplementary Figure S2). Epitope cluster analysis (http://www.iedb.org/home_v3.php) was used to assess and visualize the conservancy of the epitope regions (Supplementary Figure S2). The suitability of the selected peptide sequences for production and purification was analyzed using the peptide synthesis and proteotypic peptide analyzing tool from ThermoFisher Scientific (https://www.thermofisher.com/us/en/home/life-science/protein-biology/peptides-proteins/custom-peptide-synthesis-services/peptide-analyzing-tool.html, accessed on 20 January 2021) (not shown). The selection of peptides was based on the conducted in silico assessments for antigenicity, conservancy and suitability for the synthetic production. Finally, the selected peptides corresponded to the amino acid residues exJSRV-Env596-614, CAEV-Env860-879 and MVV-Env860-876 based on the GenBank accession numbers: AFM29008.1, NP_040942 and ALU34108.1, respectively. Customized rabbit Polyclonal Antibodies were obtained from ThermoFisher Scientific (Table 1). Immunohistochemical investigation for the three viruses was performed on all 110 lungs. PCR-positive OPA cases with typical pathomorphological lesions as well as goat synovial membrane cells (P3) infected with either MVV or CAEV served as positive controls.