Development and validation of an E2 based indirect ELISA for serological detection of porcine Getah virus

Getah virus (GETV) is an emerging mosquito borne alphavirus that has recently expanded in Chinese swine herds and causes reproductive losses and reduced growth in pigs. The absence of a standardized high throughput serological assay has limited surveillance and hindered accurate assessment of GETV circulation. In this study, we developed and validated an indirect ELISA based on recombinant full length E2 protein for detection of anti-GETV IgG in swine serum. The E2 gene from a circulating strain was expressed in Escherichia coli as inclusion bodies, followed by denaturation, redox refolding, and purification to > 90% purity. Positive and negative control sera were defined by virus neutralization. Systematic optimization identified 1 μg/mL antigen coating and 1:100 serum dilution as optimal, and the cutoff was set at OD_{450 nm} = 0.33 based on [Formula: see text] + 3SD. The assay exhibited strong analytical performance, with intra assay CVs of 3.1-5.6% and inter assay CVs of 6.3-8.8%, and diagnostic specificity and sensitivity of 94% and 98% with AUC = 0.9852. No cross reactivity was detected with sera positive for seven common porcine viruses, and positive signals remained detectable at serum dilutions up to 1:1600. Testing of 788 field sera from a commercial farm in Hubei Province revealed a seroprevalence of 56.2% indicating active GETV circulation in the herd. Collectively, these results demonstrate that the E2 based iELISA is simple, robust, and well suited for large scale serological surveillance and epidemiological investigation of GETV.