Foot-and-mouth disease (FMD) is a highly transmissible infectious disease of ungulates such as cattle, pigs, sheep, goats, deer, and water buffaloes. The causative agent, FMD virus (FMDV) is a member of the genus Aphthovirus of the family Picornaviridae (Rueckert, 1996). There are seven serotypes of the virus namely., O, A, C, Asia-1, and Southern African Territories- (SAT-) 1, 2 and 3. The genome of FMDV is ∼8.4 kb long single-stranded positive sense RNA, which is translated as a single polypeptide precursor for four structural proteins (SPs) and eight non-structural proteins (NSPs). SPs make up the capsid of the virus while the NSPs namely, Lpro, 2 A, 2B, 2 C, 3 A, 3B, 3Cpro and 3Dpol constitute the functional proteins in virus replication and assembly.

In FMD endemic countries, currently the viral infection is being controlled by vaccinating the susceptible animals with inactivated virus vaccine. For effective serosurveillance, it is essential that vaccinated animals should be differentiable from infected animals to identify FMDV infection within vaccinated populations (Barnett et al., 2015). To simplify differentiation of infected from vaccinated animals (DIVA), a vaccine needs to be free from NSPs. NSP free vaccines are relevant especially with the FMD eradication program. Though vaccine manufacturing involves steps to remove NSPs, there might be traces of residual NSPs, which could lead to the induction of antibodies, confounding with those resulting from infection. So far, different NSPs, namely, Lb, 2 C, 3 A, 3D and 3 ABC have been targeted for DIVA assays (Crowther, 2007). These tests are mostly designed to detect antibodies against the non-structural proteins after repeated immunization in animals, using ELISA based assays. Among the antibodies elicited against NSPs, antibodies against 3ABC are considered to be prominent and hence reliable in distinguishing vaccinated from infected animals (Lu et al., 2007, Mackay et al., 1998). As an alternative to detecting antibodies to NSPs in vaccinated animals, it is more desirable to have an in-process control test during vaccine formulation, to detect NSPs. Previously, filtration-assisted chemiluminometric enzyme-linked immunosorbent assay (FAL-ELISA), sandwich enzyme-linked immunosorbent assay and lateral flow assay (LFA) have been reported for detection of residual non-structural proteins (Capozzo et al., 2010, Fu et al., 2023, Kim et al., 2019, Sareyyupoglu et al., 2020). However, FAL-ELISA being sensitive to slightest of handling errors and miscalculations, requires expertise for assessment and interpretation of the results and although LFA has been established as a point-of-care assay, it requires an additional instrument for quantification of proteins (Kim et al., 2019). Therefore, in this study, we have developed an inexpensive and simple dot blot immunoassay targeting 3A and 3B proteins to detect NSPs for utilization during FMD vaccine formulation. Since FMD control program involves mass vaccination, the dot blot immunoassay could be useful in evaluating large number of vaccine batches in a short time, as a quality control tool in FMD vaccine manufacturing.