Usutu virus (USUV), one of the most neglected Old World encephalitic flaviviruses, causes epizootics among wild and captive birds and sporadic infection in humans. The dynamics of USUV spread and evolution in its natural hosts are unknown. Here, we present the phylogeny and evolutionary history of all available USUV strains, including 77 newly sequenced complete genomes from a variety of host species at a temporal and spatial scaled resolution. The results showed that USUV can be classified into six distinct lineages and that the most recent common ancestor of the recent European epizootics emerged in Africa at least 500 years ago. We demonstrated that USUV was introduced regularly from Africa into Europe in the last 50 years, and the genetic diversity of European lineages is shaped primarily by in situ evolution, while the African lineages have been driven by extensive gene flow. Most of the amino acid changes are deleterious polymorphisms removed by purifying selection, with adaptive evolution restricted to the NS5 gene and several others evolving under episodic directional selection, indicating that the ecological or immunological factors were mostly the key determinants of USUV dispersal and outbreaks. Host-specific mutations have been detected, while the host transition analysis identified mosquitoes as the most likely origin of the common ancestor and birds as the source of the recent European USUV lineages. Our results suggest that the major migratory bird flyways could predict the continental and intercontinental dispersal patterns of USUV and that migratory birds might act as potential long-distance dispersal vehicles.

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.

Mosquitoes and other arthropods may transmit medically important pathogens, in particular viruses such as West Nile virus. The presence of suitable hosts and competent vectors for those zoonotic viruses is essential for an enzootic transmission, which is a prerequisite for epidemics. To establish reliable risk projections, it is an urgent need for an exact identification of mosquito species, which is especially challenging in the case of sibling species, such as Culex. pipiens pipiens biotypes pipiens and molestus. To facilitate detection of different Culex pipiens forms and their hybrids we established a multiplex real-time PCR. Culex pipiens samples were obtained by egg raft collection and rearing until imago stage or adult sampling using CO2 baited traps and gravid traps. In total, we tested more than 16,500 samples collected all over Germany in the years 2011 and 2012. The predominant species in Germany are Culex pipiens pipiens biotype pipiens and Culex. torrentium, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes at sites where both species occur sympatrically. This report of a potentially important bridge vector for West Nile virus might have major impact in the risk projections for West Nile virus in Germany.

BackgroundThe East Asian mosquito species Aedes koreicus was recorded out of its native range for the first time in Belgium in 2008. Since then, several other European populations or single individuals have been observed throughout Europe with reports from Italy, Switzerland, European Russia, Slovenia, Germany and Hungary. The Italian population seems to be the only one that is expanding rapidly, so the Swiss population very likely derives from it.ResultsIn a surveillance program for invasive mosquito species, a single larva of Ae. koreicus was found in a cemetery vase in 2016 in the city of Wiesbaden, Germany. In the following year the finding was confirmed and an established population could be proven over an area of about 50 km2. The morphological identification of the first larva was confirmed by sequencing of a region within the nad4 sequence. A study of adult females showed that the morphological characteristics of this population are not identical to the populations from Belgium and Italy. The eggs and larvae were found together with Aedes j. japonicus in the same breeding sites and ovitraps, as well as with other indigenous mosquito species such as Culex pipiens/Culex torrentium, Aedes geniculatus and Anopheles plumbeus.ConclusionsSince the newly discovered population in Germany shows different morphological characteristics to the populations in Belgium and Italy, it seems to originate from an independent introduction. It remains unknown how the introduction took place. A further spread similar to the one in northern Italy can be assumed for the future due to similar climatic conditions.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3199-4) contains supplementary material, which is available to authorized users.

BackgroundKnowledge of the potential vector role of Culicidae mosquitoes in Germany is very scanty, and until recently it was generally assumed that they are not involved in the transmission of anthroponotic or zoonotic pathogens in this country. However, anticipated changes in the course of global warming and globalization may alter their status.MethodsWe conducted a molecular mass screening of mosquitoes for filarial parasites using mitochondrial 12S rRNA-based real-time PCR.ResultsNo parasites causing disease in humans such as Dirofilaria spp. were detected in about 83,000 mosquitoes tested, which had been collected in 2009 and 2010 in 16 locations throughout Germany. However, minimum infection rates of up to 24 per 1000 mosquitoes were revealed, which could be attributed to mosquito infection with Setaria tundra and a yet unidentified second parasite. Setaria tundra was found to be widespread in southern Germany in various mosquito species, except Culex spp. In contrast, the unidentified filarial species was exclusively found in Culex spp. in northern Baden-Württemberg, and is likely to be a bird parasite.ConclusionsAlthough dirofilariasis appears to be emerging and spreading in Europe, the absence of Dirofilaria spp. or other zoonotic filariae in our sample allows the conclusion that the risk of autochthonous infection in Germany is still very low. Potential vectors of S. tundra in Germany are Ochlerotatus sticticus, Oc. cantans, Aedes vexans and Anopheles claviger. Technically, the synergism between entomologists, virologists and parasitologists, combined with state-of-the-art methods allows a very efficient near-real-time monitoring of a wide spectrum of both human and veterinary pathogens, including new distribution records of parasite species and the incrimination of their potential vectors.

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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). USUVspecific 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.

Abstract:In general, non-native bird species may competitively exclude native species from nest sites. The potential resource conflict between a native and an introduced bird species, the native European Starling (Sturnus vulgaris) and the introduced Asian Ring-necked Parakeet (Psittacula krameri), was inferred from hole occupancy in parks of the German Upper Rhine Valley (Wiesbaden, Mannheim, Schwetzingen, Edingen-Neckarhausen), where Ring-necked Parakeets have been known to occur for 15 to 35 years. Only 2.2 % of trees (n = 3487) had cavities with an entrance diameter ≥4 cm. The number of unoccupied tree holes was high, representing 71 % of 639 recorded cavities. A niche separation corresponding to tree size and tree species preferred by both bird species could be detected. Starling preferred trees with a diameter of 0.75 ± 0.30 m, whereas Ring-necked Parakeet favoured ones with 1.16 ± 0.36 m in diameter. Both bird species were the most abundant hole-nesters in the examined cavities. Ring-necked Parakeets preferred Platanus x hispanica, which hosted 57 % of all broods for the parakeet, but did not use Acer pseudoplatanus. Starling favoured Quercus robur with 25 % of all broods, followed by A. pseudoplatanus with 19 %. P. x hispanica showed the highest numbers of cavities with 227 in total and a median of 3 holes per tree, partially produced by Ring-necked Parakeet. The largest colony-in-one-tree in this study was 9 for P. krameri and 5 for S. vulgaris. In this study, Starlings took over two nests from Ring-necked Parakeets, with one case vice versa, and Honeybees took over one active Ring-necked Parakeet nest.