A newly developed multiplex RT-PCR enables detection of blood-associated viral nucleic acids in cats.

Feline immunodeficiency virus (FIV), feline leukemia virus (FeLV), domestic cat hepadnavirus (DCHBV), felis catus gammaherpesvirus 1 (FcaGHV1), and severe fever with thrombocytopenia syndrome virus (SFTSV) are significant blood-associated pathogens impacting feline health. Because these viruses often co-circulate, rapid and simultaneous detection is essential for effective diagnosis and surveillance. A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of viral and proviral nucleic acids associated with feline blood-borne viruses. Initial analytical evaluation using spike and non-spike controls established limits of detection ranging from 1.6 copies/µl (FeLV) to 900 copies/µl (FcaGHV1 and SFTSV). No cross-reactivity with other common feline viruses was observed during analytical specificity testing. When evaluated relative to corresponding singleplex RT-PCR assays (n = 126), the assay demonstrated 100% negative percent agreement and 91.9% positive percent agreement relative to corresponding singleplex RT-PCR assays, with diagnostic agreement (κ = 0.878). Following validation, the multiplex assay was applied to 442 clinical samples (401 blood and 41 spleen samples), identifying single detections in 17.2%, and codetections in 4.3% of cases. FeLV and FIV were most prevalent, while no SFTSV was detected; furthermore, FcaGHV1-positive samples underwent additional genetic characterization. Retroviral presence significantly correlated with clinically ill, male cats older than three years. Overall, the assay demonstrated reliable multiplex detection and may serve as a useful screening tool for feline viral surveillance and clinical evaluation, although negative results should be interpreted cautiously for low-copy targets.