Fine mapping of a salmonid E2 alphavirus neutralizing epitope

Abstract

In this study, we aimed to characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of salmonid alphavirus (SAV) subtypes and widely used in several laboratories to routinely diagnose SAV. We hypothesized that the 17H23 epitope was located in the major domain B, previously identified in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs. Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23. Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV (rSAV) by using the available infectious cDNA. Ten mutant rSAVs termed A-J from E2 aa 223-236 were produced and characterized in vitro using indirect immunofluorescence assays on virus-infected cells with mAbs 17H23, 51B8 (another non-neutralizing anti-E2 mAb) and 19F3 directed against the non-structural protein nsp1. Two of the mutant rSAVs (G and H) escaped neutralization by mAb 17H23. In addition, we showed that when juvenile trout were infected by bath immersion with the rSAV mutants, some of them were either totally (D, E and G) or partially (H) attenuated. Together, the data from the in vitro and in vivo experiments indicated that the putative 17H23 amino acid sequence epitope comprised the short amino acid sequence 227 FTSDS 231 .

Effects of a DNA and multivalent oil-adjuvanted vaccines against pancreas disease in Atlantic salmon (Salmo salar) challenged with salmonid alphavirus subtype 3

“…SAV-infected cells were visualized using an indirect immunofluorescence test according to the procedure by Falk et al. [18] , but with the use of monoclonal antibody 17H23 directed against the E2 glycoprotein of SAV [19] as the primary antibody and with biotin labelled goat anti-mouse Ig and FITC-labelled streptavidin as the secondary amplification step. Standard TCID 50 end-point titers were determined by microscopic examination and calculations according to Kärber [23] .…”

Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture

“…Cells were further fixed and washed using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, San Diego, CA, USA). Following the second wash, the cells were incubated with a monoclonal antibody against E2 (mouse anti-E2 17H23) (1:1000) [ 30 ] for 60 min at room temperature, washed twice with permeabilization buffer and incubated for 1 h at room temperature with Alexa Fluor 488-conjugated anti-mouse antibody (1:400) (Molecular Probes, Life Technologies, Eugene, OR, USA). Nuclear DNA was stained with Hoechst 33,342 (ThermoFisher, Waltham, MA USA).…”

Field Evaluation of Diagnostic Test Sensitivity and Specificity for Salmonid Alphavirus (SAV) Infection and Pancreas Disease (PD) in Farmed Atlantic salmon (Salmo salar L.) in Norway Using Bayesian Latent Class Analysis

“…Briefly, a 96-well CHSE plate was inoculated with cell culture supernatants and incubated at 15°C for 10 days. After fixation in 80% acetone, 50 μl of diluted SAV-specific mouse monoclonal antibody 17H23 directed against the E2 glycoprotein (25) was added per well and incubated for 1 h, followed by subsequent incubation for 1 h with diluted secondary biotinylated goat anti-mouse IgG antibody (DAKO) before the final incubation with streptavidin-fluorescein isothiocyanate (FITC) conjugate (eBioscience). Stained cell cultures were examined on an inverted fluorescence microscope.…”