Detection of picornavirus VP2 capsid proteins using DNA aptamer

Foot-and-mouth disease (FMD) is a highly transmissible disease caused by infection with an Aphthovirus, a member of the family Picornaviridae. There are a total of 7 serotypes of foot-and-mouth disease virus (FMDV) namely serotype A, O, Asia-1, C and South African Territories (SAT) -1, -2 and -3. In Malaysia, serotypes A, O, and Asia-1 are prevalent in which Asia-1 was last detected in the 1990s whereas serotype A was last detected in 2014. In 2015, DVS Malaysia reported that 80% of the viruses detected were serotype O (WOAH, 2015) making it the serotype with the highest infection-causing serotype. The FMD virus causes illness in cloven-hoofed animals including cows, pigs, sheep, goats, and deer. FMD virus is an economically important livestock disease worldwide. This disease is endemic in Malaysia with several major outbreaks reported. In Malaysia, livestock showing symptoms of FMD undergoes a diagnostic process of sample collections, then tested for the presence of FMDV via RT-PCR, enzyme-linked immunoassay (ELISA), and virus isolation (VI) before being deemed infected livestock. In addition, immuno-based assays are sensitive to temperature, expensive, and time- consuming. In this study, the ability of 10 pre-existing aptamers that have been designed to detect FMD virus to work as a probe in the detection of FMDV was tested. Hence this study aims to develop an aptamer-based detection probe that can be used to detect FMD virus across serotypes and its free energy binding value via molecular docking. To test the ability of all 10 aptamers to detect VP2 capsid protein of different serotypes of FMD virus was predicted. A set of pre-existing aptamers that have been designed to detect FMD virus were used to test its ability to detect VP2 capsid protein of different serotypes of FMD virus by predicting its free energy binding value via molecular docking. A total of 10 aptamers were used as ligands whereas VP2 capsid protein from FMDV serotype A, and serotype O was used as the target and the docking process was performed with AutoDock Vina and Hex 8.0.0. All 10 aptamers were later synthesised with additional biotin modification at 5’ of each aptamer. In the second objective, the ability of these aptamers’ performance in web lab settings was tested on different platforms including direct enzyme-linked apta-sorbent assay (ELASA) and aptamer-based Western blot. A recombinant protein was constructed based on VP2 capsid protein of serotype O, A and Asia-1 then expressed and purified. It was later used as a target on the different platforms tested. Results showed most aptamers have positive binding with aptamer T1, T2 and T3 having the highest sensitivity. The sensitivity of each of these aptamers is at 0.5 μM of aptamer and 20 ng/ml of protein for aptamer T1, 5 ng/ml with 0.5 μM of aptamer concentration for aptamer T2 and 5 ng/ml of protein with 0.5 μM of aptamer concentration for aptamer T3. These three aptamers were then used to verify their binding to the targeted protein via aptamer-based Western blot and measuring its binding affinity via equilibrium dissociation constant (Kd). Results show all three aptamers were able to bind specifically at purified target protein having the size of 26 kDa. These aptamers also showed high binding affinity with Kd values ranging from 3.092±0.05 nM to 87.39±0.15 nM. In objective three, the ability of the aptamer to be used as a probe in the detection method was tested again with purified live FMDV grown in IB-RS2 and control FIP virus grown in CRFK cells. Almost all aptamers showed positive binding to FMDV serotype A and serotype O. However, these aptamers do not have high sensitivity when tested with purified live FMDV grown in cell culture as most aptamers were not able to show significant binding at lower virus doses. Nevertheless, the aptamers showed a slightly higher binding affinity with FMDV serotype O compared to FMDV serotype A. The lowest virus dose to be detected for serotype O was 1 x 102 TCID50/ml as compared to 1 x 104 TCID50/ml for serotype A. It can be concluded that the aptamers especially aptamer T1, aptamer T2 and aptamer T3 are the most potential candidates to be used as probes in the FMDV detection method based on its evidence when tested on different detection platforms with different types of FMDV used as target.

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