Antibody ratios against NS1 antigens of tick‐borne encephalitis and West Nile viruses support differential flavivirus serology in dogs

Flavivirus diagnostics are complicated by substantial cross-reactivity of antibodies between different flavivirus species. This is of particular importance in regions with multiple endemic flaviviruses in co-circulation. Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis, the most common infection of the central nervous system in endemic regions of Europe and Asia. Since 2018, the related West Nile virus (WNV) has spread to Germany where its geographic distribution overlaps with TBEV endemic regions. Besides humans, various animal species are susceptible to TBEV and WNV infection. To compare antibody responses against these flaviviruses and test for cross-reactivity, we developed a multi-species luciferase immunoprecipitation system antibody detection assay for several different antigens. We performed a serosurvey of 682 dogs from five different European countries to detect antibodies against TBEV and WNV. Twelve specimens were positive for TBEV NS1 only and seven for WNV NS1 only. Two specimens were reactive to both NS1 antigens and another two were equivocal for WNV NS1. Interestingly, 89.5% of positive specimens had TBEV/WNV or WNV/TBEV signal ratios of 10 to >300 between individual NS1 antigens, allowing for a clear distinction between the two viruses. The remaining 10.5% of reactive specimens showed a 5 to 10-fold difference between the two viruses and included possible dual exposures to both viruses. In contrast, equivocal samples showed low signal ratios between the NS1 antigens, suggesting unspecific reactivity. Based on this data, we found the NS1 protein to be a suitable antigen to distinguish between TBEV and WNV-specific antibodies in dogs with sensitivity and specificity similar to virus neutralization tests.