Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

Abstract

bAn amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltosebinding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection. Foot-and-mouth disease (FMD) is the most contagious viral vesicular disease that affects cloven-hoofed livestock species. FMD has significant global socioeconomic consequences, from national livestock industries suffering because of international trade restrictions to subsistence farmers suffering because of losses of stock productivity and livelihoods. Western European, North American, and Far East Asian/Pacific regions and most South American countries have official recognition of freedom-from-FMD status, with or without vaccination, by the World Organization for Animal Health (OIE). Regions in which FMD remains endemic tend to be those of lesser economic capacity, and this limits their ability to control or to eradicate the disease (1, 2). As a result, FMD remains an ongoing problem in regions in which it is endemic, and it is a persistent threat to regions that are free of the disease. Seven serotypes have been described for foot-and-mouth disease virus (FMDV), i.e., O, A, C, Asia 1, and South African Territories 1 (SAT1), SAT2, and SAT3. The disease forms produced by each serotype are clinically indistinguishable (3); similarly, FMD is clinically indistinguishable from other vesicular lesion-causing diseases, such as vesicular stomatitis, swine vesicular exanthema, and swine vesicular disease, and laboratory-based clinical diagnosis is required (4, 5). FMD infections may be mild or subclinical in ovine or caprine species, further complicating clinical diagnosis (6, 7). Exposure to one serotype does not confer cross-serotype immunity, potentially complicating diagnosis when multiple serotypes are circulating during an outbreak (8). A definitive diagnosis of FMD is possible only with laboratory testing.Conventional serological assays for FMD, including the virus neutralization test, liquid-phase blocking enzyme-linked immunosorbent assay (ELISA), and so...

Development of a chemiluminescence immunoassay using recombinant non-structural epitope-based proteins to accurately differentiate foot-and-mouth disease virus-infected and vaccinated bovines

“…DIVA tests are crucially important to serological surveillance, which demonstrate the vaccinated herds infected with or freedom from FMD (Van Dreumel et al., ). The reference FMDV DIVA test advocated by OIE detects antibodies against 3ABC NSP using ELISA and is complemented by an enzyme‐linked immunoelectrotransfer blot assay (EITB) (Bergmann et al., ).…”

“…The reference FMDV DIVA test advocated by OIE detects antibodies against 3ABC NSP using ELISA and is complemented by an enzyme‐linked immunoelectrotransfer blot assay (EITB) (Bergmann et al., ). The NSP 3ABC is the most important indicator of FMDV infection due to its high immunogenicity (Ma et al., ; Van Dreumel et al., ).However, it is not possible to completely purify the commonly employed inactivated FMD vaccine to remove all 3ABC components. Therefore, frequently vaccinated animals produce antibodies to this NSP, which results in a high false‐positive rate (Mackay, Forsyth, Davies, & Salt, ).…”

Recombinant occlusion bodies of baculovirus as carriers of a non-structural protein of foot-and-mouth disease virus

“…Wild-type (wt) AcMNPV was obtained from Pharmingen (BD Biosciences). Antibodies used include polyclonal anti-POLH (GeneTex) and monoclonal anti-3AB1 (Trotta 2009). Secondary antibodies were horseradish peroxidase-coupled (HRP) anti-mouse (Santa Cruz), anti-chicken (Bethyl) and anti-bovine (KPL).…”

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus

“…10,18 However, variable performance of these iELISAs or cELISAs using serum samples from different geographic areas has been reported, and can contribute to decision errors in disease response and control. 2,4,9,10,17,20,24,26,30,35 Therefore, developing new, validated DIVA assays that demonstrate consistent performance across all 7 FMDV serotypes and multiple FMD-susceptible species is crucial for effective control of FMD endemic and epizootic areas. We describe herein a highly specific and sensitive, <3 h assay that can serve as an important companion test during FMD outbreak response, control, and recovery when FMDV vaccination is used.…”