A dual-method assessment of West Nile and Chikungunya viruses in blood donations from Fars province, southwestern Iran.

West Nile virus (WNV) and Chikungunya virus (CHIKV) are emerging arboviral threats with potential implications for blood transfusion safety. This study investigated the prevalence of active viremia and past exposure to these viruses among blood donors in Fars province, southwestern Iran. Between December 2023 and September 2024, a cross-sectional survey enrolled 314 asymptomatic blood donors from transfusion centers in Shiraz and Jahrom. Whole blood specimens underwent RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) targeting WNV polyprotein and CHIKV envelope E1 genes. Serum samples were tested for virus-specific IgM and IgG antibodies using commercial enzyme-linked immunosorbent assays (ELISA). No confirmatory neutralization testing was performed. None of the 314 donations yielded detectable WNV or CHIKV RNA by RT-PCR. Using the rule of three for zero-event samples, the upper 95% confidence bound for true viremia prevalence was estimated at 0.96% per virus. Serological testing identified 12 donors (3.82%) with WNV IgG antibodies, while no samples tested positive for CHIKV IgG or for IgM antibodies against either virus. Among seropositive cases, 9 originated from Shiraz (4.3% site prevalence) and 3 from Jahrom (2.9% site prevalence). The majority of WNV IgG-positive donors were male (11/12, 91.7%) and engaged in outdoor occupations such as farming or manual labor (10/12, 83.3%). All 12 positive ELISA results were reproducible upon duplicate retesting. Active viremic infections with WNV or CHIKV were absent among 314 blood donors in southwestern Iran during the study period, indicating a low transfusion-transmission risk at that time within the limitations of a single cross-sectional survey. Confirmatory neutralization testing was not performed; therefore, seroprevalence estimates are presumptive. However, the detection of WNV IgG in nearly 4% of donors provides serological evidence of prior community exposure. These findings support the need for ongoing integrated arboviral surveillance incorporating human, vector, and reservoir populations, particularly among occupationally exposed groups.