Epizootic Emergence of Usutu Virus in Wild and Captive Birds in Germany

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

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

<p>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.</p

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

<p>The phylogenetic trees inferred with MrBayes are based on nucleotide sequences and of USUV strains: <b>A</b> (length: 11003 nucleotides, complete polyprotein gene) and <b>B</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.</p

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

<p>(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).</p

Phylogeography of Lassa Virus in Nigeria

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country.IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.status: publishe