Development of a lateral flow immuno-chromatic strip assay for the detection of nervous necrosis virus (NNV, RGNNV genotype)

Viral nervous necrosis (VNN), a nodaviral disease, currently represents a global threat to sustainable marine aquaculture. An efficient and rapid on-site detection method is in need for the early diagnosis of VNN. We have developed and validated a lateral flow immuno-chromatographic assay (LFIA) using the previously reported six anti-nervous necrosis virus (NNV) monoclonal antibody (mAb) for the point-of-care detection of NNV (RGNNV genotype). mAb 2B1 was used to bio-conjugate NNV antigen with colloidal gold nanoparticles, and mAb 2B11 was used to capture antigen bound colloidal gold on the test line. A downstream control line was lined with goat anti-mouse IgG to capture unbound gold nanoparticle to validate the performance. The test result can be viewed within 10 min after the application of virus culture supernatants and brain tissue lysates in sample buffer to the sample well in separate tests. The limit of detection of the LFIA test was found to be 10(5.05) TCID50/100 mu L without any cross-reactivity to other fish viruses. Field-level validation of the assay using brain tissue of RGNNV-infected and non-infected sevenband grouper (Hyporthodus septemfasciatus) showed a specificity of 100% and sensitivity of 94.92% with 96.81% diagnostic effectiveness. The developed LFIA strip test will be a useful device for the early detection of NNV in farm-level.