Serological differentiation of West Nile virus‐ and Usutu virus‐induced antibodies by envelope proteins with modified cross‐reactive epitopes

Abstract

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that belong to the Japanese encephalitis virus serocomplex within the genus Flavivirus.Due to climate change and the expansion of mosquito vectors, flaviviruses are becoming endemic in increasing numbers of countries. WNV infections are reported with symptoms ranging from mild fever to severe neuro-invasive disease. Until now, only a few USUV infections have been reported in humans, mostly with mild symptoms.The serological diagnosis and differentiation between flavivirus infections, in general, and between WNV and USUV, in particular, are challenging due to the high degree of cross-reacting antibodies, especially of those directed against the conserved fusion loop (FL) domain of the envelope (E) protein. We have previously shown that E proteins containing four amino-acid mutations in and near the FL strongly reduce the binding of cross-reactive antibodies leading to diagnostic technologies with improved specificities. Here, we expanded the technology to USUV and analyzed the differentiation of USUV-and WNV-induced antibodies in humans. IgG ELISAs modified by an additional competition step with the heterologous antigen resulted in overall specificities of 93.94% for WNV Equad and 92.75% for USUV Equad. IgM antibodies against WNV could be differentiated from USUV IgM in a direct comparison using both antigens. The data indicate the potential of the system to diagnose antigenically closely related flavivirus infections.

West Nile Virus in Germany: An Emerging Infection and Its Relevance for Transfusion Safety

“…Since the window to detect WNV RNA in plasma or cerebrospinal fluid samples is very short (2–8 days), diagnosis of clinical cases often rests on detection of WNV antibodies. IgM-capture ELISAs and IgG immunoassays such as immunofluorescence tests are highly sensitive but very specific and may show an unacceptably high rate of false positives [ 40 ]. WNV-specific serological tests have been developed [ 40 ] but are not commercially available [ 40 ].…”

“…IgM-capture ELISAs and IgG immunoassays such as immunofluorescence tests are highly sensitive but very specific and may show an unacceptably high rate of false positives [ 40 ]. WNV-specific serological tests have been developed [ 40 ] but are not commercially available [ 40 ]. IgG detection can be confirmed by a virus neutralization test to rule out serological cross-reactivity.…”

“…IgG detection can be confirmed by a virus neutralization test to rule out serological cross-reactivity. Alternatively, confirmation of a WNV infection can be achieved by NAT, e.g., from the urine or whole blood, where higher concentrations of WNV RNA are present for longer periods of time (weeks) [ 40 , 41 , 42 ].…”

Evidence of Antibodies against the West Nile Virus and the Usutu Virus in Dogs and Horses from the Southeast of France

“…Tey are most likely due to diferences in the present ecosystems (mosquitoes and migratory birds), which are the determining factors in the transmission of the faviviruses. ELISA cross-reactivity between WNV and other faviviruses is not new, as was demonstrated previously for the tick-borne encephalitis virus [26]. TBEV is a common zoonosis in central Europe and Asia but remains nonendemic in the southeast regions of France.…”

“…Due to the high specifcity rate of the serum neutralisation assay in discriminating between the possible WNV/USUV cross-reactions often occurring within ELISA [24][25][26][27], the SNT was considered the "gold standard" for defnitive diagnosis of WNV infections [28]. Terefore, the present study followed the classical methodology based on SNT on sera from ELISA-positive animals.…”