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Background: Haemosporidians (Apicomplexa, Protista) are obligate heteroxenous parasites of vertebrates and blood-sucking dipteran insects. Avian haemosporidians comprise more than 250 species traditionally classified into four genera, Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. However, analyses of the mitochondrial CytB gene revealed a vast variety of lineages not yet linked to morphospecies. This study aimed to analyse and discuss the data of haemosporidian lineages isolated from birds of the family Turdidae, to visualise host and geographic distribution using DNA haplotype networks and to suggest directions for taxonomy research on parasite species., Methods: Haemosporidian CytB sequence data from 350 thrushes were analysed for the present study and complemented with CytB data of avian haemosporidians gathered from Genbank and MalAvi database. Maximum Likelihood trees were calculated to identify clades featuring lineages isolated from Turdidae species. For each clade, DNA haplotype networks were calculated and provided with information on host and geographic distribution., Results: In species of the Turdidae, this study identified 82 Plasmodium, 37 Haemoproteus, and 119 Leucocytozoon lineages, 68, 28, and 112 of which are mainly found in this host group. Most of these lineages cluster in the clades, which are shown as DNA haplotype networks. The lineages of the Leucocytozoon clades were almost exclusively isolated from thrushes and usually were restricted to one host genus, whereas the Plasmodium and Haemoproteus networks featured multiple lineages also recovered from other passeriform and non-passeriform birds., Conclusion: This study represents the first attempt to summarise information on the haemosporidian parasite lineages of a whole bird family. The analyses allowed the identification of numerous groups of related lineages, which have not been linked to morphologically defined species yet, and they revealed several cases in which CytB lineages were probably assigned to the wrong morphospecies. These taxonomic issues are addressed by comparing distributional patterns of the CytB lineages with data from the original species descriptions and further literature. The authors also discuss the availability of sequence data and emphasise that MalAvi database should be considered an extremely valuable addition to GenBank, but not a replacement.

An amendment to this paper has been published and can be accessed via the original article.

Background: After Action Reviews (AAR) with a One Health perspective were performed in Slovenia, Italy, Serbia and Greece following a severe West Nile virus (WNV) transmission season in 2018. A protocol combining traditional techniques and organizational process analysis was developed and then implemented in each country., Results: In 2018, response to the unusually intense transmission season of WNV in Slovenia, Italy, Serbia and Greece took place through routine response mechanisms. None of the four countries declared a national or subnational emergency. We found a very strong consensus on the strengths identified in responding to this event. All countries indicated the availability of One Health Plans for surveillance and response; very high laboratory diagnostic capacity in the human, veterinary and entomology sectors and strong inter-sectoral collaboration with strong commitment of engaged institutions as critical in the management of the event. Finally, countries implementing One Health surveillance for WNV (in terms of early warning and early activation of prevention measures) consistently reported a positive impact on their activities, in particular when combining mosquito and bird surveillance with surveillance of cases in humans and equids. Recurring priority areas for improvement included: increasing knowledge on vector-control measures, ensuring the sustainability of vector monitoring and surveillance, and improving capacity to manage media pressure., Conclusions: The AARs presented here demonstrate the benefit of cross-sectoral and cross-disciplinary approaches to preparedness for West Nile virus outbreaks in Europe. In the coming years, priorities include fostering and strengthening arrangements that: enable coordinated One Health surveillance and response during WNV transmission seasons; ensure adequate laboratory capacities; strengthen risk communication; and fund longer-term research to address the knowledge gaps identified in this study.

Background: Uranotaenia unguiculata Edwards, 1913 is a species of mosquito (Diptera: Culicidae) native to central Europe. Recently a novel lineage of the West Nile virus (WNV-lineage 4c) was identified in pools of adult female Ur. unguiculata. To increase the body of knowledge about this species, various trapping methods were evaluated to determine the most efficient method for capturing adult female Ur. unguiculata., Results: Sound traps collected equivalent numbers of female Ur. unguiculata as low-hanging light-baited downdraft traps. Hosts were identified as Pelophylax lessonae and P. ridibunda (Anura: Ranidae) species group frogs from the blood found in engorged females. In addition to confirming infection by WNV-lin. 4c, a potentially integrated flavivirus sequence was detected in male mosquitoes. A novel Alphamesonivirus 1 (Nidovirales: Mesoniviridae) was found to be widespread in the Ur. unguiculata population and is herein described., Conclusions: Efficient collection methods for Ur. unguiculata for arbovirus surveillance reflect mosquito questing behavior. Uranotaenia unguiculata targets frog species which call from the water, and it is likely that the novel WNV-lin. 4c is maintained in a frog-mosquito transmission cycle. The improved trapping methods listed here will assist future studies of the vector status of Ur. unguiculata for WNV and other arboviruses.

Background: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far., Results: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites., Conclusions: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.

Background: Avian host species have different roles in the amplification and maintenance of West Nile virus (WNV), therefore identifying key taxa is vital in understanding WNV epidemics. Here, we present a comprehensive case study conducted on red-footed falcons, where host-vector, vector-virus and host-virus interactions were simultaneously studied to evaluate host species contribution to WNV circulation qualitatively., Results: Mosquitoes were trapped inside red-footed falcon nest-boxes by a method originally developed for the capture of blackflies and midges. We showed that this approach is also efficient for trapping mosquitoes and that the number of trapped vectors is a function of host attraction. Brood size and nestling age had a positive effect on the number of attracted Culex pipiens individuals while the blood-feeding success rate of both dominant Culex species (Culex pipiens and Culex modestus) markedly decreased after the nestlings reached 14 days of age. Using RT-PCR, we showed that WNV was present in these mosquitoes with 4.2% (CI: 0.9-7.5%) prevalence. We did not detect WNV in any of the nestling blood samples. However, a relatively high seroprevalence (25.4% CI: 18.8-33.2%) was detected with an enzyme-linked immunoabsorbent assay (ELISA). Using the ELISA OD ratios as a proxy to antibody titers, we showed that older seropositive nestlings have lower antibody levels than their younger conspecifics and that hatching order negatively influences antibody levels in broods with seropositive nestlings., Conclusions: Red-footed falcons in the studied system are exposed to a local sylvatic WNV circulation, and the risk of infection is higher for younger nestlings. However, the lack of individuals with viremia and the high WNV seroprevalence, indicate that either host has a very short viremic period or that a large percentage of nestlings in the population receive maternal antibodies. This latter assumption is supported by the age and hatching order dependence of antibody levels found for seropositive nestlings. Considering the temporal pattern in mosquito feeding success, maternal immunity may be effective in protecting progeny against WNV infection despite the short antibody half-life measured in various other species. We conclude that red-footed falcons seem to have low WNV host competence and are unlikely to be effective virus reservoirs in the studied region.

Background: The Asian bush mosquito, Aedes (Hulecoeteomyia) japonicus japonicus (Theobald, 1901) (Diptera: Culicidae), was first identified in Austria in August 2011 in the federal state of Styria at the border to Slovenia., Methods: Between 2011 and 2015 the spread of Ae. j. japonicus was monitored in southern, eastern and western Austrian provinces as well as in neighbouring countries by checking natural and man-made container habitats for the aquatic stages. The search concentrated around the most recent occurrence of Ae. j. japonicus and extended up to several kilometres until the subspecies could not be found anymore., Results: Between May and July 2012 the distribution area of Ae. j. japonicus was found to be extended westwards into Carinthia, and eastwards towards the federal state of Burgenland. In August 2012, the subspecies was found in Hungary, representing the first record of an invasive mosquito species in this country. In 2013 its expansion was confirmed at several sites in Austria. Additionally, between April and July 2015, the subspecies was detected in all districts of the westernmost Austrian state Vorarlberg reaching the alpine Montafon valley at the end of October 2015, at all three examined sites in southern Bavaria bordering Vorarlberg, and in the adjacent Principality of Liechtenstein, for which it also represents the first record of an invasive mosquito species. One remarkable finding of the subspecies was located close to the city of Kufstein in the lower Inn valley of the Tyrol in September 2015, which is an isolated occurrence without spatial connection to any known established population., Conclusions: Our findings demonstrate the ongoing spread of Ae. j. japonicus towards all directions within Austria and beyond. Together with the absence of supposed natural barriers, e.g. high mountain chains, at the borders of the current subspecies' distribution area in south-eastern Austria, these findings suggest a further spread to the Austrian capital Vienna and the Hungarian tourist region of Lake Balaton within the upcoming few years. The observed intrusions in western Austria represent most probably extensions of the population established and spreading in eastern Switzerland and southern Germany. The putative role of the subspecies in pathogen transmission together with its rapid spread observed argues for the implementation of comprehensive nation-wide surveillance and response preparedness.

Background: Mosquito-borne flaviviruses are a major public health threat in many countries worldwide. In Central Europe, West Nile virus (WNV) and Usutu virus (USUV), both belonging to the Japanese encephalitis virus group (Flaviviridae) have emerged in the last decennium. Surveillance of mosquito vectors for arboviruses is a sensitive tool to evaluate virus circulation and consequently to estimate the public health risk., Methods: Mosquitoes (Culicidae) were collected at South-Moravian (Czech Republic) fishponds between 2010 and 2014. A total of 61,770 female Culex modestus Ficalbi mosquitoes, pooled to 1,243 samples, were examined for flaviviruses by RT-PCR., Results: One pool proved positive for USUV RNA. Phylogenetic analysis demonstrated that this Czech USUV strain is closely related to Austrian and other Central European strains of the virus. In addition, nine strains of WNV lineage 2 were detected in Cx. modestus collected in the same reed bed ecosystem., Conclusions: This is the first detection of USUV in Cx. modestus. The results indicate that USUV and WNV may co-circulate in a sylvatic cycle in the same habitat, characterised by the presence of water birds and Cx. modestus mosquitoes, serving as hosts and vectors, respectively, for both viruses.