Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Southwest Saudi Arabia

Background Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Serological surveillance, functional neutralization testing, and virus titration via tissue culture are important for monitoring the effectiveness of vaccines and outbreaks. Objective This study aimed to increase the precision of immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing. Methods Twenty serum samples were tested using the ID.vet RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and TCID₅₀/ml was calculated using the Reed and Muench  method. VNT was performed at 24, 48, 72, and 96 hours after infection with different viral doses (100 to 100,000 TCID₅₀/ml), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the  control, protection was determined by CPE inhibition. Results ELISA revealed robust antibody signals up to a 1:32 dilution, with S/N < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration  of 10⁶. ⁵ TCID₅₀/ml. The VNT was time and dose- dependent, with good  protection obtained at low serum dilutions and viral titers, with up to 97 protective effects at 1:2–1:8 dilutions against 100–1000 TCID₅₀/ml; however, this protection decreased at higher doses and higher serum dilutions. The results of ELISA and VNT were strongly correlated in the determination at low dilutions, whereas ELISA had decreased sensitivity at high dilutions, at which VNT was still capable of detecting neutralizing activity. Conclusion This combined strategy validates that competitive ELISA  is applicable for early and medium antibody detection and VNT can functionally validate immune protection. These results provide evidence for the added value of the combination of these two methods in assessing RVFV vaccine-induced immunity and contribute to the  further interpretation of antibody–virus kinetics.