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

Abstract

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (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 18 Blackbirds (Turdus merula). 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 from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 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. 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.

Figure 1. Spatial distribution of USUV RNA positive (n = 83) and negative (n = 61) tested birds found dead in southwest Germany.

Black dot indicate birds tested positive for USUV RNA and white dot indicate USUV RNA negative birds. 79 birds were collected outside of region shown in the map.

Figure 2. Phylogenetic analysis of the novel Usutu virus (USUV) strain BH65/11-02-03 detected in a dead Blackbird from Mannheim in southwest Germany.

The phylogenetic trees inferred with MrBayes are based on nucleotide sequences and of USUV strains: A (length: 11003 nucleotides, complete polyprotein gene) and B (length: 1327 nucleotides, partial polyprotein gene). For each sequence, the GenBank accession number, strain designation, and strain origin are provided. Posterior probabilities higher than 0.5 are shown on each node. Scale bar indicates genetic distance in nucleotide substitutions per site.

Figure 3. Immunohistochemistry (IHC) of USUV-uninfected (A, C) and –infected (B, D, F and F) blackbird organs using an USUV-specific murine monoclonal antibody.

(A) USUV-uninfected blackbird brain. (B) USUV-infected blackbird brain showing a group of USUV-positive neurons (in red). (C) USUV-uninfected blackbird heart. (D) USUV-infected blackbird heart, USUV-positive cells are localized in the endocardium (in red). (E) USUV-infected blackbird liver, disseminated USUV-positive Kupffer cells (in red). (F) USUV-infected blackbird lung, disseminated USUV-positive cells (in red).