In Silico Validation of Contemporary East African Rift Valley Fever Virus Genomes Confirms the Enduring Robustness of the Bird et al. (2007) RT-qPCR Assay

Rift Valley fever virus (RVFV) is a zoonotic pathogen causing recurrent epizootics and epi-demics in East Africa. The quantitative reverse transcription PCR (RT-qPCR) assay devel-oped by Bird et al. (2007), targeting the large (L) genome segment, remains a global diag-nostic standard. However, continuous viral evolution raises concerns regarding diagnostic drift and potential assay failure. This study evaluated the contemporary robustness of this assay against East African RVFV genomes collected between 2005 and 2026. A total of 647 nucleotide records were retrieved from NCBI GenBank, from which a dataset of 193 L-segment sequences was curated based on geography, collection date, and segment an-notation. A custom sliding-window alignment algorithm quantified mismatches between the assay oligonucleotides and all valid sequences in both genomic orientations. Five se-quences initially exhibited high mismatch counts but were excluded after genomic coor-dinate mapping revealed they were partial fragments mapping outside the assay’s target region (nucleotides 4229–4723). Among the remaining 188 valid, target-spanning se-quences, 181 (96.3%) exhibited a perfect match to the forward primer, reverse primer, and TaqMan probe. Six sequences (3.2%) contained minor, biologically tolerable mismatches, and one (0.5%) showed probe-specific mismatches with intact primer binding sites. These findings confirm the enduring robustness of the Bird et al. (2007) assay against contempo-rary East African RVFV lineages, while simultaneously highlighting a critical genomic surveillance gap in Tanzania that warrants targeted infrastructural investment.