Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

Outbreaks of Foot-and-mouth disease (FMD) have resulted in tremendous economic losses. Thus, the development of a rapid and easily performed test for FMD detection is important for controlling a FMD outbreak and containing its spread. The purpose of this project is to develop a lateral flow immunochromatographic (LFI) strip test for rapid detection of FMD virus serotypes O, A and Asia 1.

Foot-and-mouth disease (FMD) is one of the world's most highly contagious animal diseases with tremendous economic consequences. A rapid and specific test for FMD diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures. This project developed a multiplex lateral flow immunochromatographic strip test (multiplex-LFI) for the rapid detection and serotyping of FMD viruses. The monoclonal antibodies (mAbs) against serotypes O, A, and Asia 1 were used as capture mAbs. The mAbs were conjugated with fluorescein, rhodamine or biotin for serotype O, A and Asia 1, respectively. The detection mAbs which consisted of a serotype-independent mAb in combination with one serotype A-specific mAb and one Asia 1-specific mAb, were each colloidal gold-conjugated. The strips used in this study contained one control line and three test lines, which corresponded to one of the three serotypes, O, A or Asia 1. The newly developed multiplex-LFI strip test specifically identified serotype O (n = 46), A (n = 45) and Asia 1 (n = 17) in all tested field isolates. The sensitivity of this strip test was comparable to the double antibody sandwich ELISA for serotypes O and A, but lower than the ELISA for serotype Asia 1. The multiplex-LFI strip test identified all tissue suspensions from animals that were experimentally inoculated with serotypes O, A or Asia 1. FMD viruses were detected in 38% and 50% of the swab samples from the lesion areas of experimentally inoculated sheep for serotypes O and A, respectively. The capability of the multiplex-LFI strip tests to produce rapid results with high specificity for FMD viruses of multiple serotypes makes this test a valuable tool to detect FMD viruses at outbreak sites.

Foot-and-mouth disease (FMD) has severe implications for animal farming which leads to considerable financial losses because of its rapid spread, high morbidity and loss of productivity. For these reasons, the use of vaccine is often favoured to prevent and control FMD. Selection of the proper vaccine is extremely difficult because of the antigenic variation within FMDV serotypes. The aim of the current study was to produce a panel of mAbs and use it for the characterization of new isolates of FMDV serotype O. A panel of FMDV/O specific mAb was produced. The generated mAbs were then characterized using the peptide array and mAb resistant mutant selection. Seven out of the nine mAbs reacted with five known antigenic sites, thus the other two mAbs against non-neutralizing sites were identified. The mAbs were then evaluated by antigenic ELISA for the detection of forty-six FMDV serotype O isolates representing seven of ten known topotypes. Isolates ECU/4/10 and HKN/2/11 demonstrated the highest antigenic variation compared to the others. Furthermore, the panel of mAbs was used in vaccine matching by antigenic profiling ELISA with O1/Manisa as the reference strain. However, there was no correlation between vaccine matching by antigenic ELISA and the gold standard method, virus neutralisation test (VNT), for the forty-six FMDV/O isolates. Nine isolates had particularly poor correlation with the reference vaccine strain as revealed by the low r₁ values in VNT. The amino acid sequences of the outer capsid proteins for these nine isolates were analyzed and compared with the vaccine strain O1/Manisa. The isolate ECU/4/10 displayed three unique amino acid substitutions around the antigenic sites 1, 3 and 4. The panel of mAbs is useful to monitor the emergence of antigenically different strains and determination of relevant antigenic site differences. However, for vaccine matching VNT remains the preferred method but a combination of VNT, antigenic profiling with a panel of mAbs and genetic sequencing would probably be more ideal for full characterization of any new outbreak isolates as well as for selection of vaccine strains from FMDV antigen banks.

The use of swine oral fluid (OF) for the detection of nucleic acids and antibodies is gaining significant popularity. Assays have been developed for this purpose for endemic and foreign animal diseases of swine. Here, we report the use of OF for the detection of virus and antibodies in pigs experimentally infected with swine vesicular disease virus (SVDV), a virus that causes a disease clinically indistinguishable from the economically devastating foot‐and‐mouth disease. Viral genome was detected in OF by real‐time reverse transcription polymerase chain reaction (RRT‐PCR) from 1 day post‐infection (DPI) to 21 DPI. Virus isolation from OF was also successful at 1–5 DPI. An adapted competitive ELISA based on the monoclonal antibodies 5B7 detected antibodies to SVDV in OF starting at DPI 6. Additionally, using isotype‐specific indirect ELISAs, SVDV‐specific IgM and IgA were evaluated in OF. IgM response started at DPI 6, peaking at DPI 7 or 14 and declining sharply at DPI 21, while IgA response started at DPI 7, peaked at DPI 14 and remained high until the end of the experiment. These results confirm the potential use of OF for SVD surveillance using both established and partially validated assays in this study.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.