Development of a cell culture-based method for detecting infectious tick-borne encephalitis virus (TBEV) in milk products

Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 10⁶ to 10⁰ TCID₅₀/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 10⁵ to 9.28 × 10¹ TCID₅₀ per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 10³ TCID₅₀/mL in raw milk samples and to 9.28 × 10¹ TCID₅₀ from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 10² TCID₅₀/2.5g and method B to 9.28 × 10³ TCID₅₀/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.