Hanna Jöst

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.

Isolation of Usutu Virus in Germany

Usutu virus (USUV) is a mosquito-borne flavivirus that emerged 2001 in Austria and caused deaths in wild birds. In Germany, 70,378 female mosquitoes were captured in 2009 and 2010 and assayed for USUV. Virus was isolated in cell culture from one pool of Culex pipiens pipiens mosquitoes trapped exclusively in August 2010 in Weinheim, Germany. Subsequent phylogenetic analysis demonstrated a close relationship between the isolated USUV strain from Germany and a USUV strain from Austria, which was detected in a dead blackbird in 2004.

Isolation and Phylogenetic Analysis of Sindbis Viruses from Mosquitoes in Germany

ABSTRACT A molecular survey of 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 demonstrated the presence of Sindbis virus (SINV) in Culex spp. and Anopheles maculipennis sensu lato. Phylogenetic analysis of the German SINV strains linked them with Swedish SINV strains, the causative agent of Ockelbo disease in humans.

European Surveillance for West Nile Virus in Mosquito Populations

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids 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.

Isolation and Phylogenetic Analysis of Batai Virus, Germany

A molecular survey including 16,057 mosquitoes captured in Southwest Germany during the summer of 2009 showed the presence of Batai virus (BATV) in Anopheles maculipennis sensu lato. Until this survey, there was no evidence for circulation of BATV in Germany. Analysis of partial S, M, and L segments showed that the sequences from all three segments were most closely related to BATV, indicating that the virus has not undergone reassortment. Phylogenetic analysis revealed a close relationship of the isolated BATV strain from Germany with strains from Slovakia, Ukraine, and Russia.

Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany

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.

Reconstruction of the Evolutionary History and Dispersal of Usutu Virus, a Neglected Emerging Arbovirus in Europe and Africa

ABSTRACT 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. IMPORTANCE Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts. Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused massive bird die-offs, mostly in Europe. There is increasing evidence that USUV appears to be pathogenic for humans, becoming a potential public health problem. The emergence of USUV in Europe allows us to understand how an arbovirus spreads, adapts, and evolves in a naive environment. Thus, understanding the epidemiological and evolutionary processes that contribute to the emergence, maintenance, and further spread of viral diseases is the sine qua non to develop and implement surveillance strategies for their control. In this work, we performed an expansive phylogeographic and evolutionary analysis of USUV using all published sequences and those generated during this study. Subsequently, we described the genetic traits, reconstructed the potential pattern of geographic spread between continents/countries of the identified viral lineages and the drivers of viral migration, and traced the origin of outbreaks and transition events between different hosts.

Usutu virus in bats, Germany, 2013.

To the Editor: Usutu virus (USUV) is an arthropod-borne flavivirus that belongs to the Japanese encephalitis serocomplex (1). USUV circulates between ornithophilic mosquito vectors (mainly Culex spp. mosquitoes) and avian amplification hosts (2). Migratory birds play a key role in the introduction of USUV into new areas (3). USUV has recently been introduced from Africa into Europe, causing epizootics among wild birds and Usutu fever in humans (4–6). The detection and isolation of USUV from different bird and mammalophilic mosquitoes during the epizootic in Germany raise questions regarding the USUV host range (2,3). Bats have been considered natural reservoir hosts of a wide diversity of viruses, including several flaviviruses (7,8). Their ability to fly and their social behavior enable efficient maintenance, spread, and evolution of viruses.

Blood donor screening for West Nile virus (WNV) revealed acute Usutu virus (USUV) infection, Germany, September 2016

Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.