Quantitative assessment of tick-borne encephalitis virus loads across hosts and vectors using digital PCR

Tick-borne encephalitis virus (TBEV) is maintained in complex natural cycles involving multiple hosts and vectors ticks. While TBEV detection in field samples is routinely performed using RT-qPCR, quantitative data on viral load across different origins remain scarce [1]. In this study, we analysed 213 TBEV-positive samples originating from ticks, rodents, goats, dogs, horses, and raw goat milk. Viral RNA copy number per microliter was determined using absolute quantification with digital PCR. Mean viral loads (viral copies/µL) after outlier elimination were: horses 6.68, ticks 3.45, rodents 4.06, goats 3.64, milk 2.80, and dogs 3.89. Statistical comparisons revealed significantly higher viral loads in horse samples compared to ticks (p = 0.0012), rodents (p = 0.0262), goat milk (p = 0.0110), and dogs (p = 0.0398). Without outlier removal, the corresponding means were higher overall: horses 31.35, ticks 8.33, rodents 8.23, goats 3.64, milk 3.57, and dogs 3.89. These findings highlight substantial variation in the TBEV copy number depending on sample origin and wide variation within a sample type. Our results further demonstrate the utility of digital PCR for precise viral quantification in diverse biological matrices, which may aid in refining diagnostic approaches and risk assessment in both veterinary and public health contexts.