Epizootic emergence of Usutu virus in wild and captive birds in Germany

Abstract

Background: Usutu virus (USUV) is an arthropod-borne, single-stranded RNA virus and belongs to the Japanese encephalitis virus group within the family Flaviviridae. USUV is closely related to West Nile virus, which killed thousands of birds in North America following its emergence in 1999. In 2001, USUV caused deaths in Blackbirds (Turdus merula) and Great Grey Owls (Strix nebulosa) in Austria. Two human cases with an USUV-related neuroinvasive illness were reported from Italy. In August 2010, USUV strain 1477 was isolated from a pool of Culex pipiens pipiens mosquitoes that were trapped in southwest Germany. In contrast, all mosquitoes trapped the year before in southwest Germany tested negative for USUV and no obvious increase in the mortality of birds was observed in southwest Germany in 2009 and 2010. Since June 2011, dead Blackbirds were frequently found in southwest Germany. Thus, our study aimed to identify the causative agent of this mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Methods: Since June 2011, 223 dead birds were collected in southwest Germany and tested for the presence of viral pathogens with real-time RT-PCRs, immunohistochemistry and isolation methods. Results: USUV RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 14 Blackbirds. USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird. Phylogenetic analysis of the German USUV strain revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Conclusion: Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.