Arboviruses in the Astrakhan region of Russia for 2018 season: The development of multiplex PCR assays and analysis of mosquitoes, ticks, and human blood sera.

The Astrakhan region of Russia is endemic for the number of arboviruses. In this paper, we describe the results of the detection of the list of neglected arboviruses in the Astrakhan region for the 2018 season. For the purpose of the study in-house PCR assays for detection of 18 arboviruses have been developed and validated using arboviruses obtained from Russian State Collection of Viruses. Pools of ticks (n = 463) and mosquitoes (n = 312) as well as 420 samples of human patients sera have been collected and analyzed. Using developed multiplex real-time PCR assays we were able to detect RNA of eight arboviruses (Crimean-Congo hemorrhagic fever virus, Dhori (Batken strain) virus, Batai virus, Tahyna virus, Uukuniemi virus, Inkoo virus, Sindbis virus and West Nile fever virus). All discovered viruses are capable of infecting humans causing fever and in some cases severe forms with hemorrhagic or neurologic symptoms. From PCR-positive samples, we were able to recover one isolate each of Dhori (Batken strain) virus and Crimean-Congo hemorrhagic fever virus which were further characterized by next-generation sequencing. The genomic sequences of identified Dhori (Batken strain) virus strain represent the most complete genome of Batken virus strain among previously reported.

Sindbis virus- a wild bird associated zoonotic arbovirus circulates in Germany.

Sindbis virus (SINV) is an arbovirus causing clinical symptoms such as arthritis, rash and fever following human infections in Fennoscandia. Its transmission cycle involves mosquito species as vectors as well as wild birds that act as natural reservoir hosts. In Germany, SINV was first time observed in 2009 in different mosquito species in the Upper Rhine valley and one year later in a hooded crow in Berlin. Recently, SINV was also detected repeatedly at various locations in Germany in the context of a mosquitoes monitoring program for arboviruses. In this study, we detected for just the second time a SINV infection in a diseased wild bird (common wood pigeon) from Central Europe. SINV was isolated by cell culture and the complete SINV genome sequence was determined. Phylogenetic analyses revealed a close affiliation to SINV genotype I with a high similarity to human isolate sequences from Finland, Sweden and Russia. The isolate was genetically distinct from the first avian isolate suggesting the circulation of at least two different SINV strains in Germany. In order to reveal the infection frequency in SINV positive mosquito regions 749 bird blood samples were assayed serologically and SINV antibodies found primarily in resident birds. SINV is therefore endemically circulating in mosquitoes in Germany, which results in occasional bird infections. No data are yet available on zoonotic transmission to humans.

Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors.

Polyarthritis and rash caused by Sindbis virus (SINV), was first recognised in northern Europe about 50 years ago and is known as Ockelbo disease in Sweden and Pogosta disease in Finland. This mosquito-borne virus occurs mainly in tropical and sub-tropical countries, and in northern Europe it is suggested to cause regularly reoccurring outbreaks. Here a seven-year cycle of SINV outbreaks has been referred to in scientific papers, although the hypothesis is based solely on reported human cases. In the search for a more objective outbreak signal, we evaluated mosquito abundance and SINV prevalence in vector mosquitoes from an endemic area in central Sweden. Vector mosquitoes collected in the River Dalälven floodplains during the years before, during, and after the hypothesised 2002 outbreak year were assayed for virus on cell culture. Obtained isolates were partially sequenced, and the nucleotide sequences analysed using Bayesian maximum clade credibility and median joining network analysis. Only one SINV strain was recovered in 2001, and 4 strains in 2003, while 15 strains were recovered in 2002 with significantly increased infection rates in both the enzootic and the bridge-vectors. In 2002, the Maximum Likelihood Estimated infection rates were 10.0/1000 in the enzootic vectors Culex torrentium/pipiens, and 0.62/1000 in the bridge-vector Aedes cinereus, compared to 4.9/1000 and 0.0/1000 in 2001 and 0.0/1000 and 0.32/1000 in 2003 Sequence analysis showed that all isolates belonged to the SINV genotype I (SINV-I). The genetic analysis revealed local maintenance of four SINV-I clades in the River Dalälven floodplains over the years. Our findings suggest that increased SINV-I prevalence in vector mosquitoes constitutes the most valuable outbreak marker for further scrutinising the hypothesized seven-year cycle of SINV-I outbreaks and the mechanisms behind.

Introduction and Dispersal of Sindbis Virus from Central Africa to Europe.

Bird-hosted viruses have the potential to be transported over large areas of the world and to be transmitted in distant geographical regions. Sindbis virus (SINV) is a mosquito-borne alphavirus that is locally amplified in a bird-mosquito enzootic cycle and distributed all over the Old World and Australia/Oceania. Sindbis virus genotype I (SINV-I) is the cause of disease outbreaks in humans in South Africa as well as in northern Europe. To trace the evolutionary history and potential strain-disease association of SINV-I, we sequenced 36 complete genomes isolated from field material in Europe, as well as in Africa and the Middle East, collected over 58 years. These were analyzed together with 30 additional published whole SINV-I genomes using Bayesian analysis. Our results suggested that SINV-I was introduced only once to northern Europe from central Africa, in the 1920s. After its first introduction to Sweden, it spread east and southward on two separate occasions in the 1960s and 1970s. Another introduction from central Africa to southern/central Europe seems to have occurred, and where these two introductions meet, one recombination event was detected in central Europe. In addition, another recombinant strain was found in central Africa, where the most divergent SINV-I strains also originated.IMPORTANCE This study shows that only a single introduction of SINV into a new geographical area is required for spread and establishment, provided that the requisite vector(s) and reservoir(s) of epizootological and epidemiological importance are present. Furthermore, we present the first report of recombination between two strains of SINV in nature. Our study increases the knowledge on new introductions and dispersal of arboviruses in general and of SINV in particular.

Evolutionary analyses of Sindbis virus strains isolated from mosquitoes in Kenya.

Sindbis virus (SINV) is a mosquito borne virus maintained in nature in a mosquito-bird cycle, with human outbreaks known to occur in Northern Europe and parts of Africa. We analyzed five SINV strains isolated in Kenya from five different mosquito species and geographic locations between 2007 and 2013. Phylogenetic relationships and evolutionary inferences were performed using maximum likelihood and Bayesian phylogenetic inference approaches. Selection analyses were carried out based on the virus envelope glycoproteins (E1, E2) and non-structural protein (nsP4) genes. Phylogenetic analysis revealed that all the Kenyan SINV isolates belonged to genotype 1 with selection analyses suggesting that SINV E1, E2 and nsP4 protein encoding genes were predominantly evolving under negative selection.

Culex torrentium Mosquito Role as Major Enzootic Vector Defined by Rate of Sindbis Virus Infection, Sweden, 2009.

We isolated Sindbis virus (SINV) from the enzootic mosquito vectors Culex torrentium, Cx. pipiens, and Culiseta morsitans collected in an area of Sweden where SINV disease is endemic. The infection rate in Cx. torrentium mosquitoes was exceptionally high (36 infections/1,000 mosquitoes), defining Cx. torrentium as the main enzootic vector of SINV in Scandinavia.

Detection and isolation of Sindbis virus from mosquitoes captured during an outbreak in Sweden, 2013.

Mosquito-borne alphaviruses have the potential to cause large outbreaks throughout the world. Here we investigated the causative agent of an unexpected Sindbis virus (SINV) outbreak during August-September, 2013, in a previously nonendemic region of Sweden. Mosquitoes were collected using carbon dioxide-baited CDC traps at locations close to human cases. The mosquitoes were initially screened as large pools by SINV-specific quantitative RT-PCR, and the SINV-positive mosquitoes were species determined by single-nucleotide polymorphism (SNP) analysis, followed by sequencing the barcoding region of the cytochrome oxidase I gene. The proportion of the collected mosquitoes was determined by a metabarcoding strategy. By using novel strategies for PCR screening and genetic typing, a new SINV strain, Lövånger, was isolated from a pool of 1600 mosquitoes composed of Culex, Culiseta, and Aedes mosquitoes as determined by metabarcoding. The SINV-positive mosquito Culiseta morsitans was identified by SNP analysis and sequencing. After whole-genome sequencing and phylogenetic analysis, the SINV Lövånger isolate was shown to be most closely similar to recent Finnish SINV isolates. In conclusion, within a few weeks, we were able to detect and isolate a novel SINV strain and identify the mosquito vector during a sudden SINV outbreak.

Isolation of sindbis virus from a hooded crow in Germany.

Sindbis virus (SINV) is an arbovirus that causes clinical symptoms, including arthritis, rash, and fever during acute human infections. In Europe, SINV outbreaks are largely restricted to northern Europe. Intrigued by the isolation of SINV from mosquitoes in southwestern Germany in 2009, we initiated a passive arbovirus-monitoring program in birds and analyzed a total of 685 samples. By this approach, we were able to detect a SINV in a Hooded Crow in Germany for the first time. It was possible to isolate SINV virus in cell cultures and even to visualize virus particles by electron microscopy. After the determination of the complete SINV genome sequence, the phylogenetic analysis revealed its close relationship to SINV genotype I sequences previously obtained from mosquitoes in Germany and Scandinavia. This first report on the isolation of viable SINV indicates the potential involvement of crows in an enzootic circulation of SINV in Germany and Central Europe.

[Complete genome characterization of the Kyzylagach virus (KYZV) (Togaviridae, Alphavirus, Sindbis serogroup) isolated from mosquitoes Culex modestus Ficalbi, 1889 (Culicinae) collected in a colony of herons (Ardeidae Leach, 1820) in Azerbaijan].

Complete genome sequencing of the Kyzylagach virus (KYZV) LEIV-65A strain isolated from the Culex modestus Ficalbi, 1889 (Culicinae), which was collected in the colony of the Ardeidae Leach, 1820 birds on the coast of Caspian sea, Kyzyl-Agach bay, in the southern part of Azerbaijan, was carried out. KYZV has high homology (about 99%) with the Chinese XJ-160 strain of the Sindbis virus (SINV) isolated from Anopheles sp. in Xinjiang Uyghur autonomic region (north-eastern China). Homologies of KYZV and XJ-160 with European SINV strains are 82% and 93% for the nucleotide and amino acid sequences, respectively (GenBank ID: KF981618). The difference between the nucleotide sequences of KYZV and Australian SINV/SW6562 strain is 19%; amino acid sequences, 12%. Since XJ-160 strain is extremely similar to KYZV, the first could be considered as the KYZV strain. The geography of the KYZV and XJ-160 isolation points and their genetic distance from the European viruses allow the KYZV to be suggested as a SINV (genotype IV) topotypic variant typical of Transcaucasia and Central Asia.

Complete coding sequence and molecular epidemiological analysis of Sindbis virus isolates from mosquitoes and humans, Finland.

Sindbis virus (SINV) is an arthropod-borne alphavirus, which causes rash-arthritis, particularly in Finland. SINV is transmitted by mosquitoes in Finland but thus far no virus has been isolated from mosquitoes. In this study, we report the isolation of the first SINV strain from mosquitoes in Finland and its full-length protein-coding sequence. We furthermore describe the full-length coding sequence of six SINV strains previously isolated from humans in Finland and from a mosquito in Russia. The strain isolated from mosquitoes (Ilomantsi-2005M) was very closely related to all the other Northern European SINV strains. We found 9 aa positions, of which five in the nsP3 protein C terminus, to be distinctive signatures for the Northern European strains that may be associated with vector or host species adaptation. Phylogenetic analyses further indicate that SINV has a local circulation in endemic regions in Northern Europe and no novel strains are frequently being introduced.

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution.

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.

Phylogeographic structure and evolutionary history of Sindbis virus.

Sindbis (SIN) virus, Alphavirus, is a mosquito-borne and bird-associated virus with large geographic distribution in the Old World. We investigated the genetic diversity of 59 SIN strains after limited sequencing of their E2 glycoprotein genes. The SIN strains showed maximal diversity of 22.2% at the amino acid (aa) level, and formed five tentative genotypes. The SIN-I genotype included strains from Europe and Africa. Strains from Australia and East Asia formed SIN-II and SIN-III with about 12% and 15% aa divergence from SIN-I. The only isolate from New Zealand was distinct, and constitutes the SIN-V genotype. Isolates from Azerbaijan and China formed genotype SIN-IV with 15.6%-19.1% aa divergence from SIN-I to III and SIN-V. Phylogenetic analyses indicated that Aura virus was present before the recombinant alphavirus lineage arose. This is consistent with a South American origin of the SIN complex, and argue for a spread in North America before reaching Asia and Australia, followed by westward radiation into Africa and Europe. High levels of sequence identities were observed for geographic regions belonging to the same north-south axis, whereas the east-west genetic exchange appears to be limited. The observed phylogeographic structure was confirmed by distinct aa patterns within two-thirds of the structural protein-coding region of SIN virus strains from Saudi Arabia, Asia, and Australia. The present geography of the five SIN genotypes and subclusters within SIN-I correlate with major bird migration patterns.

Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany.

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.

Genetic determinants of Sindbis virus strain TR339 affecting midgut infection in the mosquito Aedes aegypti.

Mosquito midgut epithelial cells (MEC) play a major role in determining whether an arbovirus can successfully infect and be transmitted by mosquitoes. The Sindbis virus (SINV) strain TR339 efficiently infects Aedes aegypti MEC but the SINV strain TE/5'2J poorly infects MEC. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of TR339 and TE/5'2J differ at two sites, E2-55 and E2-70. We have altered the TE/5'2J virus genome by site-directed mutagenesis to contain two TR339 residues, E2-55 H-->Q (histidine to glutamine) and E2-70 K-->E (lysine to glutamic acid). We have characterized the growth patterns of derived viruses in cell culture and determined the midgut infection rate (MIR) in A. aegypti mosquitoes. Our results clearly show that the E2-55 H-->Q and the E2-70 K-->E mutations in the TE/5'2J virus increase MIR both independently and in combination. TE/5'2J virus containing both TR339 E2 residues had MIRs similar to the parental TR339 virus. In addition, SINV propagated in a mammalian cell line had a significantly lower A. aegypti midgut 50 % infectious dose than virus propagated in a mosquito cell line.

The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff.

Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.

Causative agent of Pogosta disease isolated from blood and skin lesions.

Pogosta disease is a mosquito-borne viral disease in Finland, which is clinically manifested by rash and arthritis; larger outbreaks occur in 7-year intervals. The causative agent of the disease has been suspected of being closely related to Sindbis virus (SINV). We isolated SINV from five patients with acute Pogosta disease during an outbreak in fall 2002 in Finland. One virus strain was recovered from a whole blood sample and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, which also represent the first human SINV isolates from Europe. Phylogenetic analysis indicates that the Finnish SINV strains are closely related to the viral agents that were previously isolated from mosquitoes and that are related clinically similar diseases in nearby geographic areas.

Complete genomic sequence of the Australian south-west genotype of Sindbis virus: comparisons with other Sindbis strains and identification of a unique deletion in the 3'-untranslated region.

Our previous studies have shown that two distinct genotypes of Sindbis (SIN) virus occur in Australia. One of these, the Oriental/Australian type, circulates throughout most of the Australian continent, whereas the recently identified south-west (SW) genetic type appears to be restricted to a distinct geographic region located in the temperate south-west of Australia. We have now determined the complete nucleotide and translated amino acid sequences of a SW isolate of SIN virus (SW6562) and performed comparative analyses with other SIN viruses at the genomic level. The genome of SW6562 is 11,569 nucleotides in length, excluding the cap nucleotide and poly (A) tail. Overall this virus differs from the prototype SIN virus (strain AR339) by 23% in nucleotide sequence and 12.5% in amino acid sequence. Partial sequences of four regions of the genome of four SW isolates were determined and compared with the corresponding sequences from a number of SIN isolates from different regions of the World. These regions are the non-structural protein (nsP3), the E2 gene, the capsid gene, and the repeated sequence elements (RSE) of the 3'UTR. These comparisons revealed that the SW SIN viruses were more closely related to South African and European strains than to other Australian isolates of SIN virus. Thus the SW genotype of SIN virus may have been introduced into this region of Australia by viremic humans or migratory birds and subsequently evolved independently in the region. The sequence data also revealed that the SW genotype contains a unique deletion in the RSE of the 3'UTR region of the genome. Previous studies have shown that deletions in this region of the SIN genome can have significant effects on virus replication in mosquito and avian cells, which may explain the restricted distribution of this genotype of SIN virus.

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland, Australia.

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.

Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus.

Infection with alphaviruses is common in the Chinese population. Here we report the isolation of a Sindbis-like virus from a pool of Anopheles mosquitoes collected in Xinjiang, China during an arbovirus survey. This virus, designated XJ-160, rapidly produced cytopathic effects on mosquito and hamster cells. In addition, it was lethal to neonatal mice if inoculated intracerebrally. Serologically, XJ-160 reacted with and was neutralized by an anti-Sindbis antibody. Anti-XJ-160 antibodies were found in several cohorts of Chinese subjects. The complete 11626-base nucleotide sequence of XJ-160 was determined. XJ-160 has diverged significantly from the prototype Sindbis virus, with an 18% difference in nucleotide sequence and an 8.6% difference in amino acids; there are 11 deletions and 2 insertions, involving 99 nucleotides in total. XJ-160 is most closely linked to Kyzylagach virus isolated in Azerbaijan. Both belong to the African/European genetic lineage of Sindbis virus, albeit more distantly related to other members.

Mosquito-borne viruses in western Europe: a review.

Several mosquito-borne arboviruses belonging to the genera Alphavirus, Flavivirus, and Bunyavirus have been reported to occur in mosquitoes and to infect humans and other vertebrates in western Europe. These zoonotic viruses circulate in nature either in an Aedes-mammal, Anopheles-mammal, or Culex-bird transmission cycle. Infected humans normally do not contribute to the virus circulation. West Nile virus (Flavivirus) caused an outbreak of fever, malaise, pain in eyes and muscles, and headache and encephalitis in southern France during 1962-1965, and an outbreak of encephalitis with a high case-fatality rate in Romania during 1996. West Nile virus has been isolated from birds, horses, and mosquitoes in Portugal, France, the former Czechoslovakia, and Romania. These data, together with reports of antibodies to West Nile virus in birds, domestic mammals, and humans in several other countries, show virus activity in southern and central Europe. Sindbis virus (Alphavirus) caused outbreaks of fever, rash, and arthralgia in northern Europe during 1981-1982, 1988, and 1995. Two California group viruses (Bunyavirus), Tahyna virus and Inkoo virus, have been identified in western Europe. Tahyna virus causes fever and respiratory symptoms and sometimes also central nervous system involvement. It occurs in most countries of central and southern Europe, and is most common in central Europe. Inkoo virus has not been associated with disease in humans in western Europe although Russian studies indicated that it can cause encephalitis. Inkoo virus occurs in northern Europe, especially in the far north. Batai virus of the Bunyamwera-group (Bunyavirus) occurs in southern, central, and northern Europe, most frequently in central Europe. The antibody prevalence in humans generally is very low, indicating that the potential of this virus as a human pathogen is probably low in Europe. The Lednice virus (Bunyavirus) has been reported only from the former Czechoslovakia and Romania, and apparently is not transmitted to humans. In addition to the six mosquito-borne viruses documented in western Europe, there is serological evidence of infection with a Semliki Forest complex virus (Alphavirus) in central and southern Europe. Although mosquito-borne viruses presently are not considered to be the cause of major health problems in western Europe, the morbidity caused by Sindbis virus, and the morbidity and mortality caused by West Nile virus, merit further studies on the ecology, epidemiology, and medical importance of these viruses. The California group of viruses and a virus of the Semliki Forest complex may be the cause of unrecognized health problems in western Europe. Specific sampling of potential vectors for virus isolation, detailed characterization of virus strains, and the use of fully characterized strains for serological diagnosis will help to elucidate the present and future potential of mosquito-borne viruses as human pathogens in Europe.