Reliable diagnostic tests that are able to distinguish infected from vaccinated animals are a critical component of regional control programs for foot-and-mouth disease (FMD) in the affected countries. Non-structural protein (NSP) serology based on the 3ABC protein has been widely used for this purpose, and several kits are commercially available worldwide. This report presents the development of a 3ABC-antigen-based indirect ELISA, employing a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Recombinant 3ABC protein was expressed in insect cells and purified using affinity column chromatography. Using this protein, an indirect ELISA was developed and validated for the detection of NSP antibodies in serum samples collected from animals with different status of FMD. Diagnostic sensitivity and specificity were found to be 95.8 (95 % CI: 92.8-97.8) and 97.45 % (95 % CI: 94.8-99.0), respectively. The in-house ELISA compared well with the commercially available prioCHECK FMDV NS-FMD kit, with a high agreement between the tests, as determined by the kappa coefficient, which was 0.87. The in-house ELISA showed higher sensitivity for detecting vaccinated and subsequently infected animals, when compared to the reference test. Both of the tests were able to detect NSP antibodies as early as 7-8 days after experimental infection.

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Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×10 pfu/animal) or trivalent (5×10 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

In foot-and-mouth disease (FMD) control programme, liquid-phase blocking ELISA (LPBE) is widely used to assay vaccine-induced seroconversion. Currently, the assay utilizes inactivated FMD virus antigen for the detection of antibodies in serum samples. To develop a non-infectious substitute for the antigen in LPBE, we expressed the structural polypeptide of FMDV (serotype A) using a baculovirus expression system, and show that inclusion of viral 3C with reduced protease activity resulted in a higher yield of structural proteins. Structural proteins expressed in insect cells assembled into empty virus-like particles (VLPs) and showed antigenicity comparable to chemically inactivated FMDV. Screening of serum samples from FMD-vaccinated cattle showed that the test performance of VLP-LPBE had a correlation of 0.89 with conventional inactivated virus antigen LPBE. The VLP-LPBE developed here demonstrates the diagnostic application of recombinant FMDV VLPs in monitoring seroconversion following FMD vaccination.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals, with many outbreaks in the developing world. MicroRNAs (miRNAs) are non-coding RNAs that regulate antiviral defence by post-transcriptional regulation of gene expression. In this study, the host miRNA response following FMDV infection was investigated in cattle, a natural host for FMDV. A significant alteration in serum miRNA expression was detected at early stages of infection. Compared to prior to infection, on day 2 postinfection (PI), 119 miRNAs were upregulated, of which 39 were significantly upregulated (P < 0.05). Gene target prediction and pathway enrichment analysis suggested that upregulated miRNAs target innate immune signalling pathways, suggesting a homeostasis effect, possibly to limit inappropriate immune responses. Further, for the significantly upregulated miRNAs, nine miRNA recognition elements were identified in the genome sequence of FMDV serotype O, which was used for infection. The antiviral effect of four of these miRNAs was confirmed in a cell culture system. These data demonstrate that changes in miRNA expression occur during early pathogenesis, and the identification of possible miRNA targets genes could help in elucidating molecular events involved in virus-host interaction and thus could be useful in developing therapeutic strategies.