Ťahyňa virus—A widespread, but neglected mosquito‐borne virus in Europe

Ťahyňa virus (TAHV) is an orthobunyavirus and was the first arbovirus isolated from mosquitoes in Europe and is associated with floodplain areas as a characteristic biotope, hares as reservoir hosts and the mammal‐feeding mosquitoes Aedes vexans as the main vector. The disease caused by TAHV (“Valtice fever”) was detected in people with acute flu‐like illness in the 1960s, and later the medical significance of TAHV became the subject of many studies. Although TAHV infections are widespread, the prevalence and number of actual cases, clinical manifestations in humans and animals and the ecology of transmission by mosquitoes and their vertebrate hosts are rarely reported. Despite its association with meningitis in humans, TAHV is a neglected human pathogen with unknown public health importance in Central Europe, and a potential emerging disease threat elsewhere in Europe due to extreme summer flooding events.

molecular and structural differences that define the genera of the Peribunyaviridae and are characteristic of species among the orthobunyaviruses (Calisher, 1996).

Phylogenetically, TAHV shares a common ancestor with
Lumbo virus (LUMV), a CSG virus isolated from Skusea (=Aedes) pembaensis found in eastern Africa (Mozambique; Figure 1). These two viruses are a sister group to a clade that includes California encephalitis virus, La Crosse virus and Snowshoe hare virus, the latter of which has a Holarctic distribution whereas the former two are found in the Nearctic region. There is evidence of isolation by distance, as the p-distances of the L segment cds between strains from Europe and China are ~0.17, whereas p-distances within Europe are ten-fold lower. This is notable, as the p-distance of the L segment between TAHV and LUMV is also ~0.17, and the TAHV strains from China share some conserved mutations in all gene segments with LUMV. Unfortunately, only a single sequence of LUMV exists, and little is known about its ecology and distribution. As TAHV has a Palearctic distribution, stretching from Central Europe across Asia, it could have followed the historical geographic range expansion of Ae. vexans, the only member of the Aedimorphus subgenus of mosquitoes found outside the Afrotropical biogeographic region. However, as Ae. vexans has a Holarctic distribution, it is not clear why TAHV remained isolated in the Palearctic. It was observed that neutralization tests could distinguish patients previously infected with TAHV from patients previously infected with LUMV (Kunz et al., 1964), and it would be interesting to know whether similar tests could distinguish these from Chinese patients previously infected with TAHV.
Ťahyňa virus was the first arbovirus pathogenic to humans isolated from mosquitoes in Europe (the virus was named by the village of Ťahyňa in eastern Slovakia, where the virus was originally isolated from Ae. vexans mosquitoes; Bárdoš & Danielová, 1959).
After the successful isolation of TAHV from the blood of sick and hospitalized children causing influenza-like illness, considerable attention was paid to clarifying the medical significance of TAHV.
The human disease "Valtice fever" manifests as a febrile summer illness with atypical pneumonia, laryngitis and nausea, especially in children; acute neurological symptoms may occur in a relatively small percentage of patients (Šimková & Sluka, 1973;Sluka, 1969).
In the Czech Republic and Slovakia, at least 200 documented cases have been reported since the 1960s, while in subsequent decades, TAHV antibodies have been detected in 60%-80% of the elderly population, mainly in endemic areas (Bárdoš, 1977;Hubálek, Bárdoš, et al., 1979;Hubálek, Chanas, et al., 1979). Other epidemiologic studies have shown substantial regional variation in TAHV prevalence.
Fatal cases have not been reported. As TAHV is widely distributed throughout Central Europe and can cause neurological symptoms, it must be considered important for public health at present, especially since its vectors are widely distributed (WHO, 2004), and mosquito densities are closely associated with summer flooding, which is likely to occur more frequently after extreme events predicted under climate change.
In this review, we summarize the current knowledge on the history of TAHV in Europe, its geographic distribution, genome and taxonomy, transmission cycle involving mosquitoes as vectors and vertebrate hosts as reservoirs, endemic occurrence in Europe, clinical manifestations in humans and laboratory diagnosis of this neglected pathogen.

| NATUR AL ECOLOGY
Like many mosquito-borne arboviruses, TAHV is characterized by a natural focality due to the requirements for specific vectors, vertebrate hosts and ecological factors needed to maintain the transmission cycle (Figure 2; Hubálek & Rudolf, 2011). In endemic areas of Central Europe, the transmission of TAHV typically occurs in mixed or deciduous woodland forests with trees that can withstand periods of flooding (such as Salix spp., Alnus spp. and Carpinus spp.; Figure 3) and especially in flooded ecosystems (Figure 4;Bárdoš, 1974;Camp et al., 2018Camp et al., , 2021Labuda & Kožuch, 1982). Outbreaks of TAHV have been recorded in Central Europe mainly in southern Moravia (Valtice, Břeclav, Drnholec) and southern and eastern Slovakia.

Impacts
• We have limited data on the distribution and prevalence of Ťahyňa virus (TAHV) in its vectors, reservoir hosts and in the human population.
• We have limited information on the public health significance of TAHV in its natural foci in Europe. The disease it causes is neglected and underreported.
• Given global climate change, extreme precipitation events with high summer temperatures will lead to more summer flooding, providing new habitat for Ae. vexans mosquito vectors and the potential for new TAHV foci.
During these outbreaks, serological results showed that exposure to TAHV was higher in flooded and adjacent regions of large rivers, such as the Vltava, Thaya (Dyja) and Elbe, where massive populations of floodplain mosquitoes occur. The virus has been detected in various ecosystems, including the southern taiga, deciduous forests, steppes and semi-arid zones in the central and southern areas of the Russian Plain, as well as in the Pannonian plains, and in the marshland plains of the Po river basin in Italy (Aspöck & Kunz, 1971;Calzolari et al., 2022;Camp et al., 2021;L'vov et al., 1987). Serological evidence suggests that TAHV activity also extends along the Danube River, from Austria to the delta in Romania (Arcan et al., 1974;Camp et al., 2018).
In addition, the distribution of TAHV was later confirmed in Asia in various biotopes, especially in humid subtropical zones (Lu et al., 2009). Transmission activity has been observed in endemic areas with the highest dynamic infection rate in mosquitoes between July and August (Bárdoš, 1974(Bárdoš, , 1975Camp et al., 2018;Danielová & Holubová, 1977), with isolation of TAHV from mosquitoes being recorded as early as June (Aspöck & Kunz, 1971;Danielová, 1992). Although the first cases of TAHV infections in humans were observed in the second half of May, the frequency of human infections increased from June to August, and a decrease was observed in September, with the last cases occurring in October (Bárdoš, 1974).

| RE S ERVOIR HOS TS
Epizootic patterns of disease caused by arboviruses generally reflect the biology of the zoonotic reservoir (amplifying) vertebrate host and vector. Based on the results of serological studies and experimental infections, hares (L. europaeus) are the most likely amplifying host of TAHV. The evidence for this comes from comparative seroprevalence in hares versus other mammals at foci where TAHV is demonstrated to be present in host-seeking mosquitoes (Aspöck & Kunz, 1971;Bárdoš, 1975), the demonstration of Ae. vexans feeding on hares in foci where Ae. vexans mosquitoes are positive for TAHV (Camp et al., 2021), and the coincidental detection of TAHV antibodies in sentinel hares when TAHV is first detected in sampled hostseeking mosquitoes (Aspöck & Kunz, 1971;Danielová et al., 1972;Danielová & Marhoul, 1968;Málkova et al., 1974). More importantly, a study demonstrating laboratory vector and host competence was performed with wild rabbits and Ae. vexans mosquitoes (Rödl et al., 1979). The study showed that mosquitoes, which had fed on rabbits with detectable viremia, were able to transmit the virus to other rabbits. Seroconversion was revealed by a means of virus neutralization test 11 days after infection.
The principal vector(s) of TAHV are often mammalophilic, and many species of mammals have been found to be seropositive in endemic regions ( Table 1) demonstrated that many mammal species endemic to Central Europe develop a detectable viremia after subcutaneous infection with TAHV and later develop virus-specific antibodies (Rödl et al., 1977(Rödl et al., , 1978(Rödl et al., , 1979(Rödl et al., , 1987. However, to date, only rabbits and hares have been demonstrated as competent hosts. Experimental infections in primates (Macaca mulatta, M. radiata and Chlorocebus aethiops) revealed that some developed viremia and produced antibodies but did not show clinical symptoms (Šimková & Bárdoš, 1969). Following the subcutaneous infection of five chimpanzees, a febrile period of 3-to 4-day duration was observed, weakness and decreased motility were noted in three of them, and all developed specific antibodies (Bárdoš, 1974). Another group was able to show low viremia after experimental infection in only one of eight rhesus macaques, all without clinical manifestations, but most developed strong neutralizing antibody titres (Bennett et al., 2011).  (Camp et al., 2018;. Likewise, livestock are also relatively easy to sample, and TAHVreactive antibodies have been demonstrated in domestic sheep, cattle, horses and domestic pigs, also in the Czech Republic and Slovakia (Juřicová et al., 1986). Antibodies to TAHV were found in sera of wild rodents (6.5%) from Spain and Portugal (Chastel et al., 1983). Madić et al. (1993) identified antibodies to TAHV in the sera of 3 out of 15 European brown bears (Ursus arctos) in Croatia (Madić et al., 1993). The possible role of hedgehogs in the natural cycle of TAHV was investigated as a possible method for virus overwintering (LeDuc, 1979), but the results were inconclusive and other studies have suggested they play no role in the enzootic maintenance of TAHV (Lu et al., 2009).  (Calzolari et al., 2022). Thus far, only a few mosquito species meet the criteria for being considered a competent vector -i.e. one that is able to become infected with the virus and transmit the virus via saliva to a host. Evidence of vector competence of Ae. vexans, as well as mammalian host competence of rabbits, was obtained experimentally by exposing Ae. vexans to the virus by feeding on infected laboratory rabbits, demonstrating the infection, and then showing transmission to suckling mice (Danielová, 1962). Combined with the evidence from field experiments showing host selection (Camp et al., 2018) (Danielová, 1962;Danielová et al., 1966). Further experiments by Danielová et al. (1966) showed that TAHV persisted in Ae. communis, Ae. cantans, Ae. excrucians and Ae. flavescens for 8-16 days, but the transmission was not established. Danielová et al. (1966) also showed that Ae. cinereus, Ae. punctor, Culex pipiens, Culiseta annulata and Anopheles maculipennis did not contain virus 8-14 days after experimental infection (Danielová et al., 1966). However, subsequent observations and experiments reversed the status of Cs. annulata (Danielová, 1972;Danielová & Marhoul, 1968;Danielová & Minař, 1969), establishing Cs. annulata as a competent vector.

| MOSQU ITO VEC TOR S
Thereafter TAHV was isolated from Cs. annulata larvae in the early spring, suggesting that the virus had persisted over the winter in the mosquito (Bárdoš, Ryba, & Hubálek, 1975). As Ae. caspius has also been implicated as a vector, vector competence experiments should be performed (or repeated) to analyse known and potential vectors of TAHV.
The virus has also been detected in and/or isolated from Cx. modestus and An. hyrcanus. The latter is of interest, as it is a mosquito species whose range is expanding in Europe (Hubálek et al., 2014). Although prior experiments were unable to establish that An. maculipennis were capable of being infected, recent evidence suggests that these mosquitoes are also exposed to TAHV in Italy (Calzolari et al., 2022). Outside mosquitoes, two isolations of TAHV have come from biting midges Culicoides in the Bohemian-Moravian highlands (Českomoravská vysočina). This was the first isolation of a virus of the Californian serogroup from a species of the family Ceratopogonidae (Halouzka et al., 1991), and while biting midges are known to be vectors of other orthobunyaviruses, their vector competence remains to be tested.
Aedes vexans and the other putative aedine vectors are floodwater mosquitoes, laying eggs in damp soils that are prone to flooding, and emerging en masse ~2 weeks following flooding causing submersion of the eggs, and development of juvenile stages. Studies of this Holarctic mosquito species in the US have shown that eggs may remain viable for years in the soil (Horsfall, 1956), and transovarial transmission (TOT) of TAHV by the mosquitoes Ae. vexans was confirmed by Danielová and Ryba (1979). This method of vertical transmission has been shown for La Crosse virus in Ae. triseriatus, an orthobunyavirus closely related to TAHV (Reese et al., 2010;Watts et al., 1975). It has been shown -at least theoretically -that if TOT is efficient, the virus may persist in mosquito populations without the participation of an amplifying vertebrate host (Bergren & Kading, 2018;Turrell & LeDuc, 1983).
In terms of virus ecology and seasonality, the first wave of mosquitoes in the spring provides an opportunity for amplification to begin, presumably from adults infected as eggs via TOT and transstadial persistence. Floodwater mosquito species are multivoltine, and -provided a source of competent hosts -the virus may continue amplification in the environment until the late summer when most human infections have been recorded.
The incubation period lasts 7-14 days, but in some cases is as short as 3 days. Clinically, TAHV produces symptoms similar to other California serogroup orthobunyaviruses, some of which -e.g., La Crosse virus or California encephalitis virus -have received more F I G U R E 2 Ťahyňa virus circulation in a natural focus of transmission. ST, sexual (venereal) transmission; TOT, transovarial transmission; TST, transstadial transmission.

F I G U R E 3 Floodplain ecosystem situated in South Moravia (Czech Republic) -characteristic habitat for the occurrence of
Aedes vexans mosquito, primary vector of TAHV.

F I G U R E 4
Floods constitute an important prerequisite for the life cycle of Aedes vexans mosquitoes, corresponding to mass emergences of questing adult females and subsequent transmission of TAHV to humans (South Moravia, Czech Republic). clinical attention than TAHV. TAHV disease manifests mainly as a flu-like illness with a high fever that reaches a maximum of 39-40°C in patients. One of the main symptoms is severe headache, which is often accompanied by dizziness, nausea and vomiting (Demikhov, 1995;Demikhov & Chaitsev, 1995;Sluka, 1969). Other associated symptoms, typical of "encephalitic" orthobunyaviruses, include conjunctivitis, atypical pneumonia, gastrointestinal symptoms, laryngitis, anorexia and myalgia (Mittermayer et al., 1964).
Despite these virus isolations, humans appear to be incidental hosts in the transmission cycle, but specific antibodies have been detected in most European countries (Table 3), especially in adults living in areas with natural focality. Seropositivity of humans to TAHV has been reported in Austria (62%) and Hungary (50%) in the 1970s (Bárdoš & Šefčovičová, 1961;Heinz et al., 1972;Kunz et al., 1964).
Most patients with TAHV-related disease are not recognized clinically or had a less severe disease that may not be captured in case reports. Only one study conducted in Russia (Sverdlovsk region, 1994) investigated the presence of TAHV in patients with encephalitis, and seropositivity was confirmed in up to 60% of patients (Glinskikh et al., 1994). In the Austrian Alps, TAHV antibodies were detected in 0.3% of Tyrolean blood donors (Sonnleitner et al., 2014). Demikhov and Chaitsev (1995) examined blood sera from 118 patients with chronic neurological diseases by neutralization assay.

| DE TEC TI ON AND D IAG NOS IS
Basically, serum IgM antibody detection is the cornerstone of TAHV diagnosis in most clinical laboratories. The gold standard for the detection of TAHV is the virus neutralization test (Camp et al., 2018;Li et al., 2010;Lu et al., 2009;Stevanovic et al., 2022).
In addition, Juřicová (1982) used the HIT test in Czechoslovakia with formaldehyde-stabilized goose erythrocytes, which retained the ability to agglutinate with the viral antigen and showed no tendency to spontaneous agglutination. Later, the same laboratory presented a study of the haemagglutination inhibition assay for the determination of TAHV antibodies in human and animal sera (Juřicová et al., 1983). Diagnostics may also rely on the indirect immunofluorescence assay (IFA) as a reliable screening method for the detection of antibodies to "neglected" viral infections. TAHV diagnosis is also confirmed by the detection of TAHV RNA using reverse transcription polymerase chain reaction (RT-PCR). This method is useful not only to gain insight into the phylogenetic relationship between the detected viral strains but also to avoid false-positive results due to cross-contamination (Sonnleitner et al., 2014). Although TAHV infections are common, the incidence of the disease is underestimated

AUTH O R CO NTR I B UTI O N S
IR conceived and designed the study. KM, IR, JC, ZH, JM, AV and SŠ drafted the manuscript. All authors revised the final version of the manuscript, to which they contributed with critical comments.

ACK N OWLED G EM ENT
We would like to thank Radek Pečta for help with data collection.

FU N D I N G I N FO R M ATI O N
The study was financially supported by the Ministry of Health of the Czech Republic (Reg. No. NV19-09-00036).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.