Application of chimeric antigens to paper-based diagnostics for detection of West Nile virus infections of Crocodylus porosus – A novel animal test case

Abstract

Laboratory-based diagnostics such as plaque reduction neutralisation tests (PRNT) and ELISA are commonly used to detect seroconversion to flavivirus infections. However, faster, qualitative screening methods are essential for quicker diagnosis and improved patient outcomes. Lateral flow assays (LFAs) offer rapid results (5–15 mins) at the point-of-care, but few commercial flavivirus antibody detection LFAs are available.We developed an LFA using novel chimeric viral antigens produced by genetically modifying the mosquito restricted Binjari virus (BinJV) to display the outer virion proteins of pathogenic viruses like West Nile virus (WNV). The BinJV chimeric platform offers several advantages for diagnostic assay development, including rapid construction of new chimeras in response to emerging viral variants, safe, scalable antigen manufacturing, and structural indistinguishability to the wild-type pathogenic virion.To demonstrate feasibility, we applied the chimeric WNV (BinJV/WNV) antigen to LFA as the capture/test line reagent for detecting seroconversion in crocodilians to WNV – a virus affecting crocodilians across multiple continents. We confirmed the antigenic conservation of the chimera on the LFA detection surface using monoclonal antibodies. Utilising well-characterised sera (n=60) from WNV-seropositive or flavivirus-naive Australian saltwater crocodiles (Crocodylus porosus), the assay exhibited 98.8 % sensitivity and 100 % specificity, with results obtained in under 15 minutes. The LFA also accurately detected seroconversion in animals experimentally infected with WNV.This qualitative screening method can be performed both inside and outside of a laboratory, and the assay design will guide the optimisation of similar tests for detecting vector-borne viral infections in humans and other animals.