Development and validation of an actin-1 endogenous internal control for multiplex RT-qPCR surveillance of mosquito- and sand fly-borne viruses.

Internal controls are essential to ensure the reliability of molecular assays, confirming the integrity of nucleic acid extraction and testing process. This is particularly important in entomovirological surveillance, which relies on molecular testing of field-collected arthropod vectors. A key challenge is identifying a control target conserved among diverse vector species. We designed a novel primer-probe set for hydrolysis probe-based real-time PCR, targeting the actin-1 gene and assessed its performance across 21 mosquito and 9 sand fly species in Israel. The assay performance was compared to that of the ribosomal protein S17 (RpS17) assay, one of several endogenous controls reported in mosquito studies. The actin-1 RT-qPCR assay detected all mosquito and sand fly species tested, whereas the RpS17 assay covered only 11 mosquito and 4 sand fly species. Varying mosquito numbers per surveillance pool showed a negative correlation between pool size and actin-1 cycle threshold (Ct) values, confirming consistent detection across sample sizes. We further integrated actin-1 into 3 multiplex RT-qPCR assays: actin-1 + West Nile virus, actin-1 + Usutu virus, and actin-1 + representative phleboviruses: Toscana virus and Sandfly fever Sicilian virus. All assays reliably detected both actin-1 and their respective viral targets, with serial dilutions showing high efficiency and linearity. Differences between singleplex and multiplex testing were minimal showing no significant impact on assay sensitivity. Overall, the actin-1 RT-qPCR assay showed broad species coverage and reliable performance in multiplex RT-qPCR using field-collected specimens, supporting its use as an endogenous internal control in molecular arbovirus surveillance.