Development and evaluation of indirect-ELISA based on recombinant nucleoprotein for detection of Ganjam virus (GANV) specific antibodies in small ruminants

Abstract

Ganjam virus (GANV) is a tick-borne zoonotic arbovirus that has been reported in India. It is the Asian variant of Nairobi sheep disease virus (NSDV), which causes significant morbidity and mortality in small ruminants. Sero-surveillance for GANV-specific antibodies among animals is relevant for identifying risk maps and implementing control strategies. In this study, we describe the development and evaluation of a recombinant nucleoprotein (rNP) based indirect-ELISA for detecting GANV specific IgG antibodies in sheep and goat populations. A full-length NP gene encoding for recombinant NP protein (516 aa, 57 kDa) was cloned, over-expressed, and purified from Escherichia coli under denaturing conditions using affinity chromatography. The checker board titration method determined the optimum antigen concentration of 200 ng/well and a test serum dilution of 1:50 with an appropriate species-specific HRPO conjugate. Evaluation of the optimal cut-off criterion by receiver operating characteristic (ROC) and Precision-Recall analyses revealed an area under the curve (AUC) of 0.999 [95% confidence interval (CI) 0.964–1.000], positive predictive value of 1.000 and F1 score of 0.9691 against the cut off criterion of > 32 PP in sheep and goats. Repeatability of the assay revealed the intra-assay % CV of 4.61 and inter-assay % CV of 12.51, which are within the accepted criteria of 10% and 15%, respectively. In addition, the assay demonstrated a reliable detection limit of up to 1:400, with no cross-reactivity with sera specific to major sheep and goat diseases. Further, the diagnostic performance of the iELISA through Bayesian two-test analysis revealed a diagnostic sensitivity of 96% and diagnostic specificity of 93%. Furthermore, screening of randomly collected sheep ( n  = 311) and goat ( n  = 547) sera revealed a GANV specific sero-positivity of 20.58% and 31.08%, respectively, among the small ruminant population of Odisha state, India. In conclusion, the developed GANV-rNP-iELISA could be potentially used in the screening of a large number of small ruminants’ serum samples to better understand the potential risk to public health in the region.