Establishing diagnostic strategies for rustrela-virus (RusV), a recently discovered neurotropic pathogen

Rustrelavirus (RusV), one of the first known relatives of rubellavirus (RuV), is a recently dis-covered neurotropic pathogen found in placental and marsupial mammals in a zoo in North-eastern Germany. Infection with RusV resulted in encephalitis subsequent death of the dis-eased animals. Yellow-necked field mice (Apodemus flavicollis) are suspected to be the natural reservoir, since RusV has been found in these non-diseased animals at eight different locations in Mecklenburg-Vorpommern and Brandenburg so far. In this study, to gain knowledge on the zoonotic potential and to allow diagnostics of RusV, different methods were established for molecular and serological detection of RusV infec-tions. An immunofluorescence antibody test (IFAT) was established to detect antibodies of RusV-positive animals on, with structural proteins of RusV, transfected cells. Furthermore, a preliminary version of an enzyme-linked immunosorbent assay (ELISA) was used to detect RuV-positive reference sera for their cross-reactivity with RusV and ruhuguvirus (RuhV), an additional newly discovered relative of RuV. The test was not able to confirm a clear cross-reactivity between the viruses. To detect RusV-RNA two primer and probe sets, for RusV reverse transcription – quantitative polymerase chain reaction (RT-qPCR), were compared. Both detected the same RusV-positive animals but the modified set, derived from the original set, mostly with lower Cq values. In addition, to identify genetic variations of RusV, two primer pairs in the RusV-E1 gene were designed to amplify two overlapping products of together 715basepairs (bp) sequence. Se-quencing of 29 RusV-positive animals from this screening project revealed three phylogenetic clusters representing samples from Northern, Eastern or Western of the known dispersal area of the virus in Mecklenburg-Vorpommern and Northwestern Brandenburg Together the established methods allow the identification of RusV-positive animals with dif-ferent material, sera, and tissue samples, and thereby provide fundamental knowledge on the distribution and genetic variability of RusV. Furthermore, the study offers the basis to investi-gate the zoonotic potential of RusV and a possible serological cross-reactivity between RuV and RusV in the future.