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In response to the increasing number of mpox cases caused by monkeypox virus (MPXV) clade I in the African continent and the first reported travel-related clade Ib case of mpox in EU/EEA, the European Centre for Disease Prevention and Control surveyed national capability for detection and characterisation of MPXV in the EU/EEA. The results showed high level of capability for case confirmation by PCR, alongside molecular typing methods for identification of MPXV clades and/or clade I subclades within the EU/EEA.

Background and Objective: No autochthonous human cases of Japanese encephalitis (JE) have been reported to date in the European Union (EU). In this study, we assess the likelihood of Japanese encephalitis virus (JEV) introduction and transmission within the EU and propose outbreak response measures., Risk Assessment: Given the global geographical distribution of JEV, the probability of virus introduction into the EU is currently very low, with viremic bird migration being the most plausible pathway of introduction. However, this likelihood would significantly increase if the virus were to become established in the Middle East, Caucasus, Central Asia or Africa. Considering the environmental conditions that are expected to be conducive for virus circulation, there is a high likelihood of virus transmission within the EU after its introduction in environmentally suitable areas. The spread of the virus within the EU would likely occur through the movement of wild birds, pigs and mosquitoes., Mitigation: To mitigate or potentially contain the emergence of JE in the EU, early detection of both human and animal cases will be crucial., (© 2023 The Authors. Zoonoses and Public Health published by Wiley‐VCH GmbH.)

BackgroundUsutu virus (USUV) is a flavivirus with an enzootic cycle between birds and mosquitoes; humans are incidental dead-end hosts. In Europe, the virus was first detected in Italy in 1996; since then, it has spread to many European countries.AimWe aimed to report on the epidemiology, surveillance, diagnosis and prevention of USUV infection in humans, mosquitoes and other animals in the European Union/European Economic Area (EU/EEA) from 2012 to 2021.MethodsWe collected information through a literature review, an online survey and an expert meeting.ResultsEight countries reported USUV infection in humans (105 cases, including 12 [corrected] with neurological symptoms), 15 countries in birds and seven in mosquitoes. Infected animals were also found among pets, wild and zoo animals. Usutu virus was detected primarily in Culex pipiens but also in six other mosquito species. Detection of USUV infection in humans is notifiable only in Italy, where it is under surveillance since 2017 and now integrated with surveillance in animals in a One Health approach. Several countries include USUV infection in the differential diagnosis of viral encephalitis and arbovirus infections. Animal USUV infection is not notifiable in any EU/EEA country.ConclusionHuman USUV infections, mainly asymptomatic and, less frequently, with a febrile illness or a neuroinvasive disease, have been reported in several EU/EEA countries, where the virus is endemic. Climate and environmental changes are expected to affect the epidemiology of USUV. A One Health approach could improve the monitoring of its evolution in Europe.

BackgroundTravellers are generally considered good sentinels for infectious disease surveillance.AimTo investigate whether health data from travellers arriving from Africa to Europe could provide evidence to support surveillance systems in Africa.MethodsWe examined disease occurrence and estimated risk of infection among travellers arriving from Africa to Europe from 2015 to 2019 using surveillance data of arthropod-borne disease cases collected through The European Surveillance System (TESSy) and flight passenger volumes from the International Air Transport Association.ResultsMalaria was the most common arthropod-borne disease reported among travellers from Africa, with 34,235 cases. The malaria travellers' infection rate (TIR) was 28.8 cases per 100,000 travellers, which is 36 and 144 times higher than the TIR for dengue and chikungunya, respectively. The malaria TIR was highest among travellers arriving from Central and Western Africa. There were 956 and 161 diagnosed imported cases of dengue and chikungunya, respectively. The highest TIR was among travellers arriving from Central, Eastern and Western Africa for dengue and from Central Africa for chikungunya in this period. Limited numbers of cases of Zika virus disease, West Nile virus infection, Rift Valley fever and yellow fever were reported.ConclusionsDespite some limitations, travellers' health data can efficiently complement local surveillance data in Africa, particularly when the country or region has a sub-optimal surveillance system. The sharing of anonymised traveller health data between regions/continents should be encouraged.

Tick-borne encephalitis (TBE) is an infection caused by the Tick-borne encephalitis virus (TBEv) and it is common in Europe. The virus is predominantly transmitted by ticks, but other non-vectorial modes of transmission are possible. This systematic review synthesises the epidemiological impact of non-vectorial modes of TBEv transmission in Europe. 41 studies were included comprising of 1308 TBE cases. Alimentary (36 studies), handling infected material (3 studies),  blood-borne (1 study), solid organ transplant (1 study) were identified as potential routes of TBEv transmission; however, no evidence of vertical transmission from mother to offspring was reported (2 studies). Consumption of unpasteurised milk/milk products was the most common vehicle of transmission and significantly increased the risk of TBE by three-fold (pooled RR 3.05, 95% CI 1.53 to 6.11; 4 studies). This review also confirms handling infected material, blood-borne and solid organ transplant as potential routes of TBEv transmission. It is important to tracing back to find the vehicle of the viral infection and to promote vaccination as it remains a mainstay for the prevention of TBE., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Silkworm rearing activities ceased in the 1970's in several European countries. Attempts on the re-establishment of ecological and sustainable sericulture in Slovenia and Hungary are ongoing. The aim of the study was to assess the usability of locally adapted mulberry genotypes for sericulture and to estimate connections between leaf compound and silkworm performance parameters. A controlled feeding experiment of silkworms was performed to test the influence of leaves from selected trees on the growth of larvae, the health and microbiological status of larvae (e.g., gut bacterial microbiome, Bombyx mori nucleopolyhedrovirus infection), weight of cocoons and raw silk parameters. The Slovenian and Hungarian mulberry genotypes had significantly higher total protein contents, and lower total phenolic contents and differed significantly in some individual phenolics compared to the reference sericultural and fruit varieties. Significant differences were found in the contents of the macro- and microelements, namely S, Mn, Fe, and Sr. Based on correlative statistics and multivariate analysis, a combined positive influence of proteins, specific phenolics, and microelements on larval growth and silk thread parameters was predicted. The results of the study indicate that selected local Slovenian and Hungarian mulberry varieties are suitable for high-quality silk cocoon and raw silk production.

As in 2018, when a large West Nile virus (WNV) epidemic occurred, the 2022 vector season in Italy was marked by an early onset of WNV circulation in mosquitoes and birds. Human infections were limited until early July, when we observed a rapid increase in the number of cases. We describe the epidemiology of human infections and animal and vector surveillance for WNV and compare the more consolidated data of June and July 2022 with the same period in 2018.

BackgroundWest Nile virus (WNV) circulates in an enzootic cycle involving mosquitoes and birds; humans are accidental hosts.AimWe analysed human WNV infections reported between 2010 and 2018 to the European Centre for Disease Prevention and Control to better understand WNV epidemiology.MethodsWe describe probable and confirmed autochthonous human cases of WNV infection reported by European Union (EU) and EU enlargement countries. Cases with unknown clinical manifestation or with unknown place of infection at NUTS 3 or GAUL 1 level were excluded from analysis.ResultsFrom southern, eastern and western Europe, 3,849 WNV human infections and 379 deaths were reported. Most cases occurred between June and October. Two large outbreaks occurred, in 2010 (n = 391) and in 2018 (n = 1,993). The outbreak in 2018 was larger than in all previous years and the first cases were reported unusually early. The number of newly affected areas (n = 45) was higher in 2018 than in previous years suggesting wider spread of WNV.ConclusionReal-time surveillance of WNV infections is key to ensuring that clinicians and public health authorities receive early warning about the occurrence of cases and potential unusual seasonal patterns. Human cases may appear shortly after first detection of animal cases. Therefore, public health authorities should develop preparedness plans before the occurrence of human or animal WNV infections.

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.

In the last decade in Hungary and the neighbouring countries, West Nile Neuroinvasive Disease (WNND) has been caused in dramatically increasing numbers by lineage 2 West Nile Virus (WNV) strains both in horses and in humans. The disease in this geographical region is seasonal, so vaccination of horses should be carefully scheduled to maintain the highest antibody titres during outbreak periods. The objective of this study was to characterise the serum neutralising (SN) antibody titres against a lineage 2 WNV strain in response to vaccination with an inactivated lineage 1 vaccine (Equip® WNV). Thirty-two seronegative horses were enrolled in the study, 22 horses were allocated to the vaccinated group and 10 retained as unvaccinated controls. Horses were vaccinated according to the product's vaccination guidelines. A primary vaccination of two doses administered 28 days apart was initiated approximately 5 months before the WNV outbreak season, followed by a booster vaccination one year later. Blood samples were collected during a 2-year period to monitor production of SN antibodies against lineage 1 and the enzootic lineage 2 WNV strain. Mean antibody titres against lineage 1 WNV were significantly higher (P ≤ 0.05) in the vaccinated group compared to the control group at all-time points after the primary dose of vaccination. Similarly, mean antibody titres against lineage 2 WNV were significantly higher (P ≤ 0.05) in the vaccinated group compared to the control group at all time-points except at 6 months after the primary vaccination. SN antibody titres were significantly higher against lineage 1 than lineage 2 at all-time points. According to the results, vaccination with an inactivated lineage 1 vaccine induces antibodies against both WNV lineages 1 and 2 strains up to 2 years after booster vaccination, but in those geographical regions where lineage 2 strains are responsible for seasonal outbreaks, a booster vaccination should be considered earlier than 12 months after primary vaccination., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rabies is enzootic in over one hundred countries worldwide. In the European Union/European Economic Area (EU/EEA), the vast majority of human rabies cases are travellers bitten by dogs in rabies-enzootic countries, mostly in Asia and Africa. Thus, EU/EEA travellers visiting rabies enzootic countries should be aware of the risk of being infected with the rabies virus when having physical contact with mammals. They should consider pre-exposure vaccination following criteria recommended by the World Health Organization and if unvaccinated, immediately seek medical attention in case of bites or scratches from mammals. As the majority of the EU/EEA countries are free from rabies in mammals, elimination of the disease (no enzootic circulation of the virus and low number of imported cases) has been achieved by 2020. However, illegal import of potentially infected animals, mainly dogs, poses a risk to public health and might threaten the elimination goal. Additionally, newly recognised bat lyssaviruses represent a potential emerging threat as the rabies vaccine may not confer protective immunity. To support preparedness activities in EU/EEA countries, guidance for the assessment and the management of the public health risk related to rabies but also other lyssaviruses, should be developed.

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.

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics.

Usutu virus (USUV), a mosquito-borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass-mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV-related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT-PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV-associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future., (© 2019 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Lednice virus (LEDV) has been detected in Culex modestus mosquitoes in several European countries within the last six decades. In this study, phylogenetic analyses of the complete genome segments confirm that LEDV belongs to the Turlock orthobunyavirus ( Orthobunyavirus , Peribunyaviridae ) species and is closely related to Umbre, Turlock, and Kedah viruses.

Five different compounds were tested for their in vitro inhibitory effect against RABV multiplication in mouse neuroblastoma (N2A) cell line. N2A cells were infected with the fixed RABV strain CVS-11 one hour prior to adding antivirals or their respective combinations. The infectious titre of RABV as well as the quantity of viral RNA was determined in the cell culturing medium after 48 h. All five tested compounds (mouse interferon (IFN)-α and -β, ribavirin, favipiravir (T-705) and sorafenib) reduced viral replication in a concentration-dependent manner: IFN-β and sorafenib both provided 73.71% relative inhibition of viral replication in the highest non-cytotoxic concentration, while ribavirin caused 48.07%, IFN-α caused 44.87% and favipiravir caused 35.25% relative inhibition, respectively. When applied in combination, their antiviral activity was not synergistic, but a pronounced inhibition was detected when IFN-β was combined with sorafenib, ribavirin, or favipiravir. The highest antiviral effect was caused by the combination of IFN-β and sorafenib (77.19% relative inhibition). In other combinations there was an antagonistic effect detected in the reduction of viral replication. The results demonstrate that these compounds can be promising candidates for a potential combination treatment of rabies, noting that some combinations are not favourable in vitro, which makes thorough in vivo studies necessary., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recent studies demonstrated that inhibitors of pro-inflammatory molecular cascades triggered by rabies infection in the central nervous system (CNS) can enhance survival in mouse model and that certain antiviral compounds interfere with rabies virus replication in vitro. In this study different combinations of therapeutics were tested to evaluate their effect on survival in rabies-infected mice, as well as on viral load in the CNS. C57Bl/6 mice were infected with Silver-haired bat rabies virus (SHBRV)-18 at virus dose approaching LD 50 and LD 100 . In one experimental group daily treatments were initiated 4 h before-, in other groups 48 or 96 h after challenge. In the first experiment therapeutic combination contained inhibitors of tumour necrosis factor-α (infliximab), caspase-1 (Ac-YVAD-cmk), and a multikinase inhibitor (sorafenib). In the treated groups there was a notable but not significant increase of survival compared to the virus infected, non-treated mice. The addition of human rabies immunoglobulins (HRIG) to the combination in the second experiment almost completely prevented mortality in the pre-exposure treatment group along with a significant reduction of viral titres in the CNS. Post-exposure treatments also greatly improved survival rates. As part of the combination with immunomodulatory compounds, HRIG had a higher impact on survival than alone. In the third experiment the combination was further supplemented with type-I interferons, ribavirin and favipiravir (T-705). As a blood-brain barrier opener, mannitol was also administered. This treatment was unable to prevent lethal consequences of SHBRV-18 infection; furthermore, it caused toxicity in treated mice, presumably due to interaction among the components. In all experiments, viral loads in the CNS were similar in mice that succumbed to rabies regardless of treatment. According to the findings, inhibitors of detrimental host response to rabies combined with antibodies can be considered among the possible therapeutic and post-exposure options in human rabies cases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

BackgroundDuring the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.AimsWe summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.MethodsEvery patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.ResultsUntil the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.ConclusionCareful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

The present study reports the first complete genome sequence analysis of West Nile virus (WNV) strains isolated from brain samples from raptors. The results prove the circulation of closely related WNV lineage II strains in central Europe and genetic analysis revealed seven amino acid substitutions in structural (PrM 3 , E 159 and E 231 ) and in non-structural (NS1 109 , NS5 259 , NS5 310 and NS5 600 ) proteins. Observed amino acid substitutions Phe 3 and Ser 231 were common only within the lineage VII Koutango strain isolated from Rhipicephalus guilhoni tick in Senegal. Further research could reveal whether these substitutions influence the biological properties of WNV, including virulence and neuroinvasiveness.

Objective: The aim of the study was to evaluate the seroprevalence of West Nile virus (WNV) among the variable population of Eastern Slovakia., Methods: A serologic survey was conducted using 464 serum samples. The basic demographic, epidemiologic and clinical information was obtained for each serum sample at the time of specimen collection. The presence of antibodies against WNV was investigated using a commercial enzyme-linked immunosorbent assay (ELISA). All the ELISA positive samples were further analysed by a neutralization test with WNV and Usutu virus., Results: Three serum samples (0.65%) from the participants (N = 464) were considered positive for antibodies to WNV. A 29-year-old female was repeatedly exposed to mosquito bites working as a shepherdess and participating in many outdoor activities. Two other females (61 and 76 years old) were treated at the Department of Neurology due to monoparesis of the upper extremity, vertigo; both had a significant epidemiological history with frequent tick and mosquito bites and stay in an endemic region., Conclusions: Although there was no evidence of WNV infection in the Slovak Republic, the epidemiological situation in the neighbouring countries warrants vigilance and appropriate measures, including the introduction of specific diagnostic tools into clinical practice. The constant monitoring of birds and mosquitoes also seems necessary.

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.

In central Europe, at least three flaviviruses circulate among vectors and vertebrate hosts. West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses maintained in the nature by enzootic cycle between mosquitoes and birds. Tick-borne encephalitis virus (TBEV) is a flavivirus causing annual human cases in Slovakia. The aim of this study is the prevalence assessment of flavivirus infections in horses (n = 145) and birds (n = 109) by enzyme-linked immunosorbent assay (ELISA) and confirmation by neutralization test (VNT). WNV antibodies have been detected in 11.7% of tested horses and 11.9% of tested birds and confirmed in 6.9% of horse and 9.2% of bird samples. None of the WNV seropositive or dubious horses had WNV IgM (ELISA), and none of the tested horses had USUV neutralizing antibodies. Autochthonous WNV infections have been confirmed in 16.7% of horses without international travelling history. Most of them were from western Slovakia with known endemic WNV transmission. An autochthonous WNV infection in a horse from highland area of Kremnické vrchy (central Slovakia) with unknown data of WNV circulation and without travelling history was detected. TBEV antibody was detected in 6.2% of horses and in 3.4% has been confirmed. In two horses, WNV and TBEV infection could not be distinguished. Confirmed WNV seropositive were eight raptors showing nonspecific signs or suffering from trauma, one white stork, and one house sparrow. The sparrow was caught in a locality in eastern Slovakia, where WNV RNA had been previously detected in sparrows. USUV neutralizing antibodies were present in pooled sample from four Eurasian great tits. Because of insufficient volume, TBEV VNT was not carried out in birds. Results further prove the endemicity of WNV and other vector-borne flaviviruses in natural and accidental hosts in Slovakia, giving better insight in flavivirus epidemiology in European countries in general.

Usutu virus (USUV, Flaviviridae) was first reported in Europe in Austria in 2001, where it caused wild bird (mainly blackbird) mortality until 2005. Since 2006 no further USUV cases were diagnosed in the country. However, the virus emerged in other European countries (Hungary, Italy, Switzerland, Spain, Germany and the Czech Republic) between 2005 and 2011. In 2016, widespread USUV-associated wild bird mortality was observed in Germany, France, Belgium and the Netherlands. In this study, we report the results of passive monitoring for USUV in Austria and Hungary between 2010 and 2016. In Hungary, USUV caused sporadic cases of wild bird mortality between 2010 and 2015 (altogether 18 diagnosed cases), whereas in summer and autumn 2016 the number of cases considerably increased to 12 (ten blackbirds, one Eurasian jay and one starling). In Austria, USUV was identified in two blackbirds in 2016. Phylogenetic analyses of coding-complete genomes and partial regions of the NS5 protein gene revealed that USUVs from Hungary between 2010 and 2015 are closely related to the virus that emerged in Austria in 2001 and in Hungary in 2005, while one Hungarian sequence from 2015 and all sequences from Hungary and Austria from 2016 clustered together with USUV sequences reported from Italy between 2009 and 2010. The results of the study indicate continuous USUV circulation in the region and exchange of USUV strains between Italy, Austria and Hungary.Emerging Microbes &Infections (2017) 6, e85; doi:10.1038/emi.2017.72; published online 11 October 2017.

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.

Between July and August 2017, seven of 12,047 blood donations from eastern Austria, reacted positive to West Nile virus (WNV) in the cobas test (Roche). Follow-up investigations revealed Usutu virus (USUV) nucleic acid in six of these. Retrospective analyses of four blood donors diagnosed as WNV-infected in 2016 showed one USUV positive. Blood transfusion services and public health authorities in USUV-endemic areas should be aware of a possible increase of human USUV infections.

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.

West Nile virus (WNV) mainly infects birds, horses and humans. Outcomes of the infection range from mild uncharacteristic signs to fatal neurologic disease. The main objectives of the present study were to measure serum IgG and IgM antibodies in naturally exposed and vaccinated horses and to compare results of haemagglutination inhibition test (HIT), enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralisation test (PRNT). Altogether 224 animals were tested by HIT for WNV antibodies and 41 horses were simultaneously examined by ELISA and PRNT. After primary screening for WNV antibodies, horses were vaccinated. Samples were taken immediately before and 3-5 weeks after each vaccination. McNemar's chi-squared and percent agreement tests were used to detect concordance between HIT, ELISA and PRNT. Analyses by HIT confirmed the presence of WNV antibodies in 27/105 (26%) naturally exposed horses. Sera from 57/66 (86%) vaccinated animals were positive before the first booster and from 11/11 (100%) before the second booster. HIT was less sensitive for detecting IgG antibodies. We could detect postvaccination IgM in 13 cases with IgM antibody capture ELISA (MAC-ELISA) and in 7 cases with HIT. WNV is endemic in Hungary and regularly causes natural infections. Protective antibodies could not be measured in some of the cases 12 months after primary vaccinations; protection is more reliable after the first yearly booster. Based on our findings it was not possible to differentiate infected from recently vaccinated horses using MAC-ELISA. HIT cannot be used as a substitute for ELISA or PRNT when detecting IgG, but it proved to be a useful tool in this study to gain statistical information about the tendencies within a fixed population of horses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Six kea (Nestor notabilis) in human care, naturally infected with West Nile virus (WNV) lineage 2 in Vienna, Austria, in 2008, developed mild to fatal neurological signs. WNV RNA persisted and the virus evolved in the birds' brains, as demonstrated by (phylo)genetic analyses of the complete viral genomes detected in kea euthanized between 2009 and 2014. WNV antibodies persisted in the birds, too. Chronic WNV infection in the brain might contribute to the circulation of the virus through oral transmission to predatory birds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, we determined the sequence of the coding region of an avian bornavirus detected in a blue-and-yellow macaw (Ara ararauna) with pathological/histopathological changes characteristic of proventricular dilatation disease. The genomic organization of the macaw bornavirus is similar to that of other bornaviruses, and its nucleotide sequence is nearly identical to the available partial parrot bornavirus 5 (PaBV-5) sequences. Phylogenetic analysis showed that these strains formed a monophyletic group distinct from other mammalian and avian bornaviruses and in calculations performed with matrix protein coding sequences, the PaBV-5 and PaBV-6 genotypes formed a common cluster, suggesting that according to the recently accepted classification system for bornaviruses, these two genotypes may belong to a new species, provisionally named Psittaciform 2 bornavirus.

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.

Background: Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained., Methods: To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively., Results: Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro., Conclusions: Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use., (Copyright © 2015 John Wiley & Sons, Ltd.)

We sequenced West Nile viruses (WNVs) from Turkey and found close relationships to WNV lineage 1 strain ArB310/67 from the Central African Republic, distinct from other WNVs circulating in the Mediterranean Basin, eastern Europe, and the Middle East. These findings suggest independent introductions of WNV strains from Africa to the Middle East.

West Nile virus (WNV) is an increasing public health concern in Europe with numerous human cases. A total of 23,029 female mosquitoes were tested for a variety of mosquito-borne flaviviruses and orthobunyaviruses supposedly endemic in Southern Transdanubia, Hungary, in the frames of a large-scale surveillance between 2011 and 2013. WNV nucleic acid was detected in a single pool containing Uranotaenia unguiculata mosquitoes. Sequence- and phylogenetic analyses for two different regions (NS5 and E) of the viral genome showed that the novel Hungarian WNV strain was different from other previously described WNV lineages. These findings may indicate the presence of a putative, novel lineage of WNV in Europe. Our results also indicate that U. unguiculata mosquito may become relevant species as a potential vector for West Nile virus in Europe.