2.2. Rabies Virus Micro-Neutralization Test

The micro-neutralization test was developed to test smaller volumes of test serum making it an ideal comparison to the traditional rapid fluorescent focus inhibition test (RFFIT) [33]. The micro- neutralization test was conducted following the protocol outlined in Smith et al. [33] using the CVS-11 RABV variant. All serum samples were screened at 1:10 dilution and positive sera were run to end-point. A cut off titer of 1:10 (0.1 IU/mL) was chosen based on the value for 50% neutralization of the challenge virus in accordance with previous studies [33,34].

The procedure for performing the micro-neutralization test involves using four-well Teflon coated slides to perform serial dilutions while employing a humidity chamber throughout the procedure to avoid any evaporation. Minimum essential medium (MEM) (12 µL) was added to each well of each slide and 3 µL of each test serum was serially diluted. Standard rabies immunoglobulin (SRIG) was used as positive control serum in preparing the positive control slide [35]. CVS-11 was prepared for the working dilution at 50 FFD₅₀ (50% fluorescent focus forming doses) per mL and 12 µL of this working dilution of virus, was added to each well of each test slides as well as to the positive control slide and the back-titration slide. The slides were incubated in the humidity chamber at 37 °C with 0.5% CO₂ for 90 min. After completion of this incubation period, 24 µL of mouse neuroblastoma cells (MNA) were added to each well, equivalent to 1.4 × 10⁴ cells per well. Slides were again incubated at 37 °C for 20 h with 0.5% CO₂ then fixed with acetone and stained with FITC-anti-rabies immunoglobulin (Fujirebio Diagnostics, Malvern, PA, USA) before being observed for the presence of fluorescent foci with a fluorescence microscope. The Reed-Muench method [36] was used to calculate the endpoint titer, which was converted to international units per millilitre (IU/mL) based on comparison to SRIG diluted at 2 IU/mL.