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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.

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.

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: 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.)

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.

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.

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 a widely distributed mosquito-borne flavivirus. WNV strains are classified into several genetic lineages on the basis of phylogenetic differences. Whereas lineage 1 viruses are distributed worldwide, lineage 2 WNV was first detected outside of Africa in Hungary in 2004. Since then, WNV-associated disease and mortality in animal and human hosts have been documented periodically in Hungary. After the first detection of WNV from a pool of Culex pipiens mosquitoes in 2010, samples were collated from several sources and tested in a 2-year monitoring program. Collection areas were located in the Southern Transdanubium, in northeastern Hungary, in eastern Hungary, and in southeastern Hungary. During the 2 years, 23,193 mosquitoes in 645 pools were screened for WNV virus presence with RT-PCR. Three pools were found positive for WNV in 2011 (one pool of Ochlerotatus annulipes collected in Fényeslitke in June, one pool of Coquillettidia richiardii collected in Debrecen, Fancsika-tó, in July, and one pool of Cx. pipiens captured near Red-Footed Falcon colonies at Kardoskút in September). The minimal infection rate (MIR=proportion of infected mosquitoes per 1000 mosquitoes) of all mosquito pools was 0.25, whereas the MIR of infected species was 2.03 for O. annulipes, 0.63 for C. richiardii, and 2.70 for C.x pipiens. Molecular data have demonstrated that the same lineage 2 WNV strain has circulated in wild birds, horses, humans, and mosquitoes in Hungary since 2004. Mosquito-based surveillance successfully complemented the ongoing, long-term passive surveillance system and it was useful for the early detection of WNV circulation.

We report the detection and isolation of four almost identical strains of West Nile virus (WNV) lineage 2from Culex modestus mosquitoes collected at three fish ponds in South Moravia, Czech Republic, during August 2013. Phylogenetic analysis demonstrated that the Czech WNV strains isolated are closely related to Austrian, Italian and Serbian strains reported in 2008,2011 and 2012, respectively. Our findings show the current northernmost range of lineage 2 WNV in Europe.

For the first time outside sub-Saharan Africa, a lineage 2 West Nile virus (WNV) emerged in Hungary in 2004. It caused sporadic cases of encephalitis in goshawks (Accipiter gentilis), other predatory birds, and in mammals. As a consequence, a surveillance program was initiated in Hungary and in Austria, which included virological, molecular, serological and epidemiological investigations in human beings, birds, horses, and mosquitoes. The virus strain became endemic to Hungary, however only sporadic cases of infections were observed between 2004 and 2007. Unexpectedly, explosive spread of the virus was noted in 2008, when neuroinvasive West Nile disease (WND) was diagnosed all over Hungary in dead goshawks and other birds of prey (n=25), in horses (n=12), and humans (n=22). At the same time this virus also spread to the eastern part of Austria, where it was detected in dead wild birds (n=8). In 2009, recurrent WND outbreaks were observed in Hungary and Austria, in wild birds, horses, and humans in the same areas. Virus isolates of both years exhibited closest genetic relationship to the lineage 2 WNV strain which emerged in 2004. As we know today, the explosive spread of the lineage 2 WNV in 2008 described here remained not restricted to Hungary and Austria, but this virus dispersed further to the south to various Balkan states and reached northern Greece, where it caused the devastating neuroinvasive WND outbreak in humans in 2010., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The emergence of lineage 2 strains of WNV in Europe as a cause of clinical disease and mortality in horses raised the question whether the existing WNV vaccines, all based on lineage 1 strains, protect against circulating lineage 2 strains of WNV. In the present paper we have determined the level of cross protection provided by the recombinant ALVAC(®)-WNV vaccine in a severe challenge model that produces clinical signs of WNV type 2 disease. Ten horses were vaccinated twice at 4 weeks interval with one dose of the ALVAC-WNV vaccine formulated at the minimum protective dose. A further 10 horses served as controls. Two weeks after the second vaccination, all horses were challenged intrathecally with a recent neurovirulent lineage 2 strain of WNV. The challenge produced viraemia in 10 out of 10 and encephalitis in 9 out of 10 control horses. Three horses had to be euthanized for humane reasons. In contrast, none of the vaccinated horses developed WNV disease and only 1 vaccinated horse became viraemic at a single time point at low titre. The prevalence of WNV disease and viraemia were significantly lower in the vaccinated horses than in the control horses (P<0.0001 for both). Based on these results, the ALVAC-WNV vaccine will provide veterinarians with an effective tool to control infections caused by lineage 1 and 2 strains of WNV., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The spread of lineage 2 West Nile virus (WNV) from sub-Saharan regions to Europe and the unpredictable change in pathogenicity indicate a potential public and veterinary health threat and requires scientific awareness., Objectives: To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses., Animals: Seventeen horses with neurologic signs., Methods: Information regarding signalment, clinical signs, and outcome was obtained for each animal. Serology was performed in 15 cases, clinicopathological examination in 7 cases, and cerebrospinal fluid was collected from 2 horses. Histopathology was carried out in 4 horses, 2 of which were assessed for the presence of WNV in their nervous system., Results: WNV neutralizing antibody titers were between 10 and 270 (median, 90) and the results of other serological assays were in agreement with those of the plaque reduction neutralization test. Common signs included ataxia, weakness, asymmetric gait, muscle tremors, hypersensitivity, cranial nerve deficits, and recumbency. Twelve animals survived. Amplicons derived from the infection-positive specimens allowed molecular characterization of the viral strain., Conclusions and Clinical Importance: From our results, we conclude that this outbreak was caused by a lineage 2 WNV strain, even though such strains often are considered nonpathogenic. Neurological signs and survival rates were similar to those reported for lineage 1 virus infections. The disease occurrence was not geographically limited as had been the typical case during European outbreaks; this report describes a substantial northwestern spread of the pathogen., (Copyright © 2011 by the American College of Veterinary Internal Medicine.)

We conducted a complete genome analysis of a West Nile virus detected in Culex pipiens mosquitoes during a severe outbreak of human West Nile disease in Greece 2010. The virus showed closest genetic relationship to the lineage 2 strain that emerged in Hungary in 2004; increased virulence may be associated with amino acid substitution H249P.

In the southeast of Hungary a sparrow hawk (Accipiter nisus) and several goshawk (Accipiter gentilis) fledglings succumbed to encephalitis manifesting as an acute neurological disease during the summers of 2004 and 2005. Both years the causative agent was identified as a lineage 2 West Nile virus. This is the first description of clinical, pathological and immunohistochemical findings of infection caused by a neuroinvasive, lineage 2 West Nile virus and the first evidence of its circulation in continental Europe.

Two different West Nile virus (WNV) strains caused lethal encephalitis in a flock of geese and a goshawk in southeastern Hungary in 2003 and 2004, respectively. During the outbreak in geese, 14 confirmed human cases of WNV encephalitis and meningitis were reported in the same area. Sequencing of complete genomes of both WNV strains and phylogenetic analyses showed that the goose-derived strain exhibits closest genetic relationship to strains isolated in 1998 in Israel and to the strain that emerged in 1999 in the United States. WNV derived from the goshawk showed the highest identity to WNV strains of lineage 2 isolated in central Africa. The same strain reemerged in 2005 in the same location, which suggests that the virus may have overwintered in Europe. The emergence of an exotic WNV strain in Hungary emphasizes the role of migrating birds in introducing new viruses to Europe.

The authors investigated an outbreak of West Nile Fever characterized by severe neurological symptoms and death in a flock of 3600 6-week-old geese. Ataxia, intermittent torticollis and opisthotonus, incoordination, rhythmic side-to-side movement of the head, wriggling of the neck and abnormal head position were features of the disease. Death occurred within 4 to 5 days after the clinical signs appeared. The average daily mortality was 5 to 15, reaching 14% (in total) over a period of 6 weeks. There were no consistent gross pathological lesions, but in a few cases yellowish-grey foci of 3 to 6 mm in diameter were observed on the surface or transection of the brain. Histopathology revealed perivascular lymphohistiocytic infiltration and glia cell proliferation in the brainstem, cerebellum, cortex and spinal cord as well as degeneration of neural fibres in the spinal cord. In addition to the lesions caused by the West Nile Virus in the brain, characteristics of circovirus infection such as lymphocyte depletion, vacuolization and basophilic intra-cytoplasmic inclusion bodies containing circovirus-like particles were seen by light and electron microscopy in the cloacal bursa. West Nile Virus infection was confirmed by reverse transcriptase-polymerase chain reaction amplification of virus-specific nucleic acid from tissue samples of the brain. Based on the nucleotide sequence analysis of the polymerase chain reaction products, 99% identity was found on the tested NS5 region with the IS-98 ST1 strain isolated from a stork in Israel in 1998, and with West Nile Virus stains emerging in the USA in 1999. Using an indirect fluorescent antibody test, high antibody titres against the virus were detected in the serum samples submitted from the affected flock. In selected sera this was confirmed by neutralization antibody test as well.

Abstract: An update on the mosquito-borne flavivirus species including certain subtypes, as listed in the Eighth Report of the International Committee on Taxonomy of Viruses, is given. Special emphasis is placed on viruses which have been shown to cause diseases in animals, and viruses for which no pathogenicity has been proven yet. Several recent examples (Usutu virus and lineage-2 West Nile virus in central Europe, Zika virus in Micronesia) have shown that sources providing information on such scientifically largely neglected viruses are valuable tools for scientists and public health officials having to deal with such disease emergences. Furthermore the effects of global warming will lead to introduction of competent mosquito vectors into temperate climate zones and will increase efficiency of viral replication in less competent vector species. This, facilitated by rising global travel and trade activities, will facilitate introduction and permanent establishment of mosquito-borne viruses, some of which may become of public health or veterinary concern, into novel environments, e.g. industrialized countries worldwide. [Copyright &y& Elsevier]