Rapid Detection of Getah Virus Antibodies in Horses Using a Recombinant E2 Protein-Based Immunochromatographic Strip

Abstract

The prevalence and impact of Getah virus (GETV) are significant concerns in China. GETV can infect a wide range of animals, including horses, pigs, sheep, cattle, birds, and humans, resulting in substantial losses in the livestock and agricultural industries. GETV infection can cause the development of ulcers and inflammation in the mouth and gums of horses, which result in pain and discomfort and lead to symptoms such as reduced appetite, drooling, and difficulty chewing. As a result, there is a pressing need for efficient and rapid disease diagnosis methods. However, the currently available diagnostic methods have limitations in terms of operational time, equipment, and the experience of the individuals using them. In this study, a rapid, specific, and sensitive detection method was developed using a colloidal gold-based immunochromatographic strip (ICS) for the detection of antibodies against GETV in horses. To prepare the ICS, the antigen domain of the E2 glycoprotein of GETV was expressed using the Escherichia coli expression system after analysis with DNAstar v7.1 software. The nitrocellulose membrane was coated with rE2 protein or SPA to form the test line and control line, respectively. After optimizing the reaction conditions, the sensitivity, specificity, and repeatability of the strip were verified. The results showed that the test strip had a detection limit of up to 1:320 dilutions for GETV-positive serum, with no cross-reactivity observed with other equine-susceptible pathogens such as equine arteritis virus (EAV), equine herpesvirus-1 (EHV-I), equine infectious anemia virus (EIAV), equine influenza virus (EIV), African horse sickness virus (AHSV), and Japanese encephalitis virus (JEV). Furthermore, the ICS exhibited a concordance rate of 94.0% when testing 182 clinical serum samples compared to the virus neutralization test. Overall, this ICS diagnosis method will be an effective tool for the rapid detection of GETV in the field.

Keywords: Getah virus; colloidal immunochromatographic strip; horse; rapid detection.

Figure 1

Schematic diagram of the immunochromatographic strip (ICS) test for detecting GETV antibodies in horse. (A) Structure of the ICS. (B) Interpretation of the results using ICS. A positive sample shows two red bands on the membrane strip; a negative sample shows only one band on the control line. If there is no colored band at all or there is only one colored band on the test line, the test is invalid. C, control line; T, test line; S, sampling hole.

Figure 2

Design, preparation, and identification of recombinant protein rE2. (A) Amino acid sequence of the E2 protein of Getah virus strain SH05-6 was obtained from GenBank (accession number: EU015066.1) and included three functional domains. Antigenic domains of Getah virus E2 glycoprotein were analyzed using the Protean program of DNAstar v7.1. Colored boxes indicate domains A, B, and C, which were predicted in reference to those of Chikungunya virus [22]. (B) Production and identification of recombinant protein rE2.M: protein marker. Lane 1: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of purified rE2 (33 kDa) expressed by vector pCold I. Lanes 2 and 4: Western blotting analysis of the E. coli-containing pCold using anti-His monoclonal antibody (Lane 2) and GETV-positive horse serum (Lane 4). Lanes 3 and 5: Western blotting analysis of the E. coli-containing pCold-E2 using anti-His monoclonal antibody (Lane 3) and GETV-positive horse serum (Lane 5).

Figure 3

Sensitivity and specificity testing of the ICS assay. (A) Sensitivity of the ICS. GETV-positive serum was diluted from 1:10 to 1:1280 to determine the sensitivity of the ICS. The detection limit of the test strip was up to 1:320 dilutions for the GETV-positive serum. Neg: negative horse serum. (B) Specificity of the ICS. Sera positive for different horse viruses (EHV1, EAV, EIV, EIAV, AHSV, and JEV) were used to evaluate the specificity of the ICS. The ICS did not cross-react with other viral-positive sera.