Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep

Abstract

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

Introduction

Bluetongue (BT), an infectious, non-contagious and arthropod-borne virus disease of ruminants belonging to the family Reoviridae and the genus Orbivirus (Lefkowitz et al., 2018), culpable for an estimated annual loss of 3 billion US dollars. Clinical signs of BT are variable and usually detected in fine wool breeds of sheep and the white-tailed deer (Odocoileus virginianus), which include fever, facial oedema, haemorrhages and ulceration on the oral mucosa and coronitis (Sperlova and Zendulkova, 2011). Existence of multiple serotypes (n = 28) with limited immunological cross reactivity have made the BT control a difficult task. The disease has worldwide distribution and in India, based on serological evidence, it is considered to be enzootic with as many as 21 serotypes reported to be prevalent (Prasad et al., 2009) and newer previously non-existing serotypes (Susmitha et al., 2012; Krishnajyothi et al., 2016; Hemadri et al., 2017), are isolated frequently. The disease is serologically monitored by both structural (VP1-VP7) and non-structural protein (NSP) based immuno-assays. Currently, for the detection of antibodies to bluetongue, VP7 based ELISAs are in vogue world over (Afshar et al., 1992; Cloete et al., 1994; Pathak et al., 2008; Hosamani et al., 2011; Gandhale et al., 2010). There are few variations of this test; some used truncated, while others used full length recombinant protein, with varying sensitivity. Among several NS proteins of BT virus, NS1 and NS3 have been the choice as candidate antigens in the development of immuno-diagnostics (Barros et al., 2009; Anderson et al., 1993) especially as suitable DIVA candidate. However, the difficulty in production/purification of either native or recombinant full length NS proteins of BTV in sufficient quantity and quality, due to inherent structural complexities have hindered the applicability of these proteins as immunodiagnostic reagents. In our previous study (Mohanty et al., 2019), the above problems were circumvented by constructing a novel NS1 and NS3 fusion gene (~1302 bp), that coded for NS1 N-terminus (₁M to G₂₅₂ aa) and N- and C-termini of NS3 protein. The construction process involved removal of two hydrophobic domains along with intervening variable central domain (₁₁₈A to A₁₈₂ aa) of NS3 and C-terminus of NS1 before fusion of the fragments. Using this strategy, we could clone, over-express the NS1-NS3 fusion gene in a prokaryotic expression system to produce a hybrid protein that was efficiently purified using a novel protocol (Mohanty et al., 2019). Herein, we extrapolated the work further, so as to come up with a diagnostic (ELISA), which could be used for surveillance of BTV infection and we hope that it will serve as a tool for differentiating infected from vaccinated animals once the marker vaccine becomes available. Additionally, to the best of our knowledge, for the first time, two NSPs have been brought together in one format for surveillance of bluetongue. Therefore, it was expected that, the current test under the study could be able to detect antibodies from a wider spectrum of infected animals as compared to the test relying on a single NS protein.