Quantitative profiling of tick-borne encephalitis virus in hosts and vectors using digital PCR in cell culture models

Tick-borne encephalitis virus (TBEV) is a major neurotropic virus circulating in Lithuania, maintained through a natural cycle between ticks and vertebrate hosts. Although RT-qPCR is widely used for virus detection in field samples, quantitative data on viral replication, particularly from infected cell cultures, remain limited. To investigate this aspect, we analyzed 139 TBEV-positive samples originating from ticks (n = 16), rodents (n = 69), dogs (n = 32), horses (n = 7), goats (n = 4), and raw goat milk (n = 11). Viral RNA copy number per microliter was determined using absolute quantification with digital PCR. Additionally, positive samples were cultured through ten consecutive passages in three different cell lines - MARC145, Vero, and N2a, followed by digital PCR to evaluate their capacity for viral amplification. Mean viral loads measured in native samples (viral copies/µL) were: horse blood 6.14 (95% CI 0 – 12.59), ticks 3.36 (95% CI 1.95 – 4.77), rodent tissues 6.72 (95% CI 3.82 – 9.63), goat blood 2.56 (95% CI 0 – 5.52), goat milk 4.54 (95% CI 2.28 – 6.8), and dog blood 4.25 (95% CI 2.76 – 5.74). Inferential analysis confirmed a statistically significant passage-dependent increase only in MARC-145 cells (slope = 0.20, 95% CI 0.12 – 0.27), (p < 0.001), although overall viral load remained very low. Furthermore, comparison of viral loads before and after cell culture revealed a strong increase only in the vector group (p < 0.001), while the domestic and synanthropic groups did not change significantly. These findings highlight the utility of digital PCR for precise viral quantification in diverse biological matrices, aiding improvement of diagnostic approaches and risk assessment in veterinary and public health contexts. Moreover, despite being rarely used for TBEV culture in practice, MARC-145 cells could be a promising first-choice system for experimental applications. However, due to very low viral copy numbers, amplification in cell culture is not a suitable method for sample preparation when a high viral load is required.