Entwicklung von diagnostischen Methoden zum Nachweis von europäischen humanpathogenen Arboviren
Development of diagnostic methods for the detection of European humanpathogenic arboviruses
A majority 50-70% of aseptic meningo-encephalitis cases is caused by unknown aetiological agents.
In the course of this work a cytometric bead array (CBA) for simultaneous detection of antibodies against the following 15 neglected European arboviruses was developed: Batai virus (BATV), Bhanja virus (BHAV), Erve virus (ERVEV), Eyach virus (EYAV), Inkoo virus (INKV), Palma virus (PALV), Sandfly fever virus Naples (SFNV), Sandfly fever virus Sicilian (SFSV), Sandfly fever virus Turkish (SFTV), Tahyna virus (TAHV), tick borne encephalitis virus (TBEV), Toscana virus (TOSV), Tribec virus (TRBV), Uukuniemi virus (UUKV) and West Nile fever virus (WNV). These viruses belong to different virus families and are transmitted by ticks, mosquitoes or sandflies.
Arboviral proteins (N for Bunyaviruses, NS1 for Flaviviruses and VP6/VP7 for Reoviruses) were expressed, purified and coupled to different, optically distinguishable subpopulations of magnetic beads. The CBA was validated, the sensitivity was determined and cross reactivity was reviewed by fluorescent detection via MAGPIX-System. To compare the sensitivity of our CBA with other known detection methods, Immunfluorescence tests (IFT) on infected VeroB4 and VeroE6 cells were performed with the corresponding viruses.
The CBA cut-off was determined based on results from 45 uninfected reference human sera. 111 samples from patients with symptoms of aseptic meningitis or aseptic meningo-encephalitis of unknown aetiology from the Upper Rhine Valley areas and the Black Forest in Southwest-Germany were tested. 42 (38%) showed positive results. 31 (28%) showed signs of acute infection (IgM: positive / IgG: negative or IgM: positive / IgG: positive) for BHAV/PALV, ERVEV, EYAV, TRBV, TBEV, INKV/TAHV, WNV, SFNV/TOSV, SFSV/SFTV. 11 (10%) showed signs of passed infection (IgM: negative / IgG: positive) for SFNV/TOSV, INKV/TAHV, EYAV, TRBV. Most of these results were confirmed by IFT.
The established CBA allows the simultaneous detection of antibodies against up to 15 European arboviruses in patient material and can help to assess the impact of these viruses as aetiological agents of aseptic meningitis and aseptic meningo-encephalitis cases of unknown origin.