Associations between the presence of specific antibodies to the West Nile Virus infection and candidate genes in Romanian horses from the Danube delta.

The West Nile virus (WNV) is a mosquito-borne flavivirus causing meningoencephalitis in humans and animals. Due to their particular susceptibility to WNV infection, horses serve as a sentinel species. In a population of Romanian semi-feral horses living in the Danube delta region, we have analyzed the distribution of candidate polymorphic genetic markers between anti WNV-IgG seropositive and seronegative horses. Thirty-six SNPs located in 28 immunity-related genes and 26 microsatellites located in the MHC and LY49 complex genomic regions were genotyped in 57 seropositive and 32 seronegative horses. The most significant association (pcorr < 0.0002) was found for genotypes composed of markers of the SLC11A1 and TLR4 genes. Markers of five other candidate genes (ADAM17, CXCR3, IL12A, MAVS, TNFA), along with 5 MHC class I and LY49-linked microsatellites were also associated with the WNV antibody status in this model horse population. The OAS1 gene, previously associated with WNV-induced clinical disease, was not associated with the presence of anti-WNV antibodies.

West Nile virus (lineage 2) detected for the first time in mosquitoes in Southern Bohemia: new WNV endemic area?

Here we report the first detection of lineage 2 of neuroinvasive West Nile virus (WNV-2) in mosquitoes collected in a fishpond area of the Trebon Basin in southern Bohemia during the 2018 mosquito season. A total of 6790 mosquito females belonging to the Culex modestus, Culex pipiens, and Coquillettidia richiardii species were investigated in 136 pools, and WNV RNA was detected in two of them. The WNV strain shares genetic homology with other WNV-2 strains isolated in southern Moravia as well as with those causing outbreaks in southern and central Europe. The results highlight the need for entomological surveillance of pathogenic arboviruses even in areas not yet affected (WNV-free areas). The South Bohemian Region (in addition to southern Moravia) is becoming another risk zone of autochthonous occurrence of West Nile fever in the Czech Republic.

West Nile virus (lineage 2) in mosquitoes in southern Moravia - awaiting the first autochthonous human cases.

Here we report repeated detection of lineage 2 West Nile virus (WNV-2) from Culex modestus and Cx. pipiens mosquitoes collected at fishponds in the Lednice-Valtice Area during the mosquito seasons 2015 and 2016. The WNV strains recovered share genetic homology with WNV strains isolated during an extensive monitoring in 2013 as well as with strains circulating in southern and central Europe at the same time. Repeated detection of WNV indicates its establishment in the area and also warns infection specialists and epidemiologists about possible emergence of human cases or even outbreaks of West Nile fever in the region.

Francisella tularensis prevalence and load in Dermacentor reticulatus ticks in an endemic area in Central Europe.

A total of 7778 host-seeking adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks were examined for the prevalence of Francisella tularensis holarctica (Thiotrichales: Francisellaceae) in a natural focus of tularaemia in the floodplain forest-meadow ecosystem along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) between 1995 and 2013. Ticks were pooled (10 specimens per pool) and their homogenates inoculated subcutaneously in 4-week-old specific pathogen-free mice. Dead mice were sectioned, their spleens cultivated on thioglycollate-glucose-blood agar and impression smears from the spleen, liver and heart blood were Giemsa-stained. Sixty-four pools were positive for F. tularensis: the overall minimum infection rate (MIR) was 0.82%. Overall MIRs for the 4714 female and 3064 male D. reticulatus examined were 0.89 and 0.72%, respectively; MIRs fluctuated across years between 0.0 and 2.43%. The estimated bacterial load in infected ticks varied from 0.84 to 5.34 log10 infectious F. tularensis cells per tick (i.e. from about seven to 220 000 cells). Ticks with low loads were more prevalent; more than 1000 infectious cells were detected in 24 ticks (0.3% of all ticks and 37.5% of infected ticks). Monitoring of D. reticulatus for the presence and cell numbers of F. tularensis may be a valuable tool in the surveillance of tularaemia.

[Human hantavirus diseases - still neglected zoonoses?]. / Onemocnení cloveka zpusobená hantaviry - stále opomíjené zoonózy?

Hantavirus disease is the most common rodent-borne viral infection in the Czech Republic, with a mean annual incidence of 0.02 cases per 100 000 population and specific antibodies detected in 1% of the human population. Four hantaviruses (Puumala, Dobrava-Belgrade, Tula, and Seewis) circulate in this country, of which Puumala virus (responsible for a mild form of hemorrhagic fever with renal syndrome called nephropathia epidemica) and Dobrava-Belgrade virus (causing haemorrhagic fever with renal syndrome) have been proven to cause human disease. The aim of this study is to provide a comprehensive review of the hantaviruses occurring in the Czech Republic, based on the literature published during the past three decades, including their geographical distribution and clinical symptoms. The recent detection of Tula virus in an immunocompromised person as well as reports of Seoul virus infections in Europe highlight the possible emergence of neglected hantavirus infections in the foreseeable future.

West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North?

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.

Seroprevalence study of hepatitis E virus infection in two districts of the Czech Republic.

The aim of this work was to retrospectively examine serum samples from 230 people living in two districts in the Czech Republic in year 1989 (Znojmo and Jihlava) for the IgG antibodies against hepatitis E virus (HEV). For examination, commercial ELISA kit DIA.PRO was used and 13 seropositive persons were found, i.e. 5.7% of those examined. The seropositivity rate was higher in women (6.8%) than in men (3.7%), but the difference was not statistically significant. The age of examined persons did not reveal a significant effect on the seropositivity rate: mean age of seropositive subjects was 38.0 years while that of seronegative persons was 39.4 years.

Usutu virus in blackbirds (Turdus merula), Czech Republic, 2011-2012.

The central European lineage of Usutu virus was isolated from a blackbird (Turdus merula), which was found dead in the city of Brno, Czech Republic, in 2011. The virus RNA was detected in two other dead blackbirds in Brno during 2012.

Isolation of Tahyna Virus (California Encephalitis Group) From Anopheles hyrcanus (Diptera, Culicidae), a Mosquito Species New to, and Expanding in, Central Europe.

Two strains of Tahyna virus (TAHV; Orthobunyavirus, Bunyaviridae) were isolated from 4,568 (92 pools) female Anopheles hyrcanus Pallas (Diptera, Culicidae) mosquitoes collected on the fishponds in South Moravia (Czechland, central Europe) during July-August 2013. This is the first isolation of TAHV from An. hyrcanus in Europe. An. hyrcanus is a species new to Czechland since 2007; its population density was very high in the year 2013 at these ponds. The virus isolation procedure was based on intracerebral inoculation of newborn mice; moreover, the positive pools were also tested by polymerase chain reaction and found to contain TAHV RNA. An. hyrcanus, feeding preferentially on mammals including humans, may be a new potential vector for TAHV in Europe.

An invasive mosquito species Aedes albopictus found in the Czech Republic, 2012.

Between July and September 2012, seventeen larvae of the invasive mosquito species Aedes (Stegomyia) albopictus (Skuse) were discovered using 60 ovitraps at four study sites alongside two main road exits in South Moravia, Czech Republic. This is the first report of imported Ae. albopictus in the Czech Republic. The findings highlight the need for a regular surveillance programme to monitor this invasive species throughout western and central Europe.

[Examination of mosquitoes collected in southern Moravia in 2006-2008 tested for arboviruses]. / Vysettení komáru na prítomnost arboviru na jizní Morave v letech 2006-2008.

The main goals of the study were to carry out virus isolation attempts on Vero cell cultures from mosquitoes collected in southern Moravia (Breclav district, Czech Republic) and to identify the isolates using a microtiter virus neutralization test. A total of 9.742 female mosquitoes belonging to 13 species were examined, and three viral strains were isolated, all from the mosquitoes collected in 2006: two of these isolates were identified as Tahyna Orthobunyavirus (both obtained from Aedes vexans mosquitoes) and one was West Nile flavivirus (obtained from Aedes rossicus mosquitoes). Ae. rossicus might be a new vector for West Nile virus.

Mosquito (Diptera: Culicidae) fauna in an area endemic for West Nile virus.

Mosquito collections with CDC light traps using dry ice and pigeon-baited traps were carried out in south Moravia (Czech Republic) from April to October in 2007 and 2008 at two study sites. In 2007, 11 two-day captures were carried out in two-week intervals, and 1,490 female mosquitoes of nine species were caught. In 2008, 15 two-day trappings of mosquitoes were carried out: 6,778 females of 22 species of mosquitoes were trapped. The results showed marked differences in abundance and species composition of mosquitoes between both study sites and between the trapping methods. In the floodplain forest ecosystem of the Soutok study area, Aedes vexans predominated. The species composition in the Nesyt study site was more varied and the most common species was Culex pipiens. At the latter study site, Anopheles hyrcanus (var. pseudopictus) and Uranotaenia unguiculata, mosquito species with largely southern Eurasian distribution, were repeatedly demonstrated. The largest capture of mosquitoes was in traps with CO2 placed at a height 1 m above the ground. The capture of mosquitoes in the pigeon-baited traps as well as in the traps with CO2 placed in the canopy of trees was markedly lower in both study sites, with the predominant species being Culex pipiens.

Mosquito (Diptera: Culicidae) surveillance for arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Tahyna viruses in Central Europe.

Six viral isolates were obtained from 23,243 female mosquitoes (examined in 513 pools) belonging to 16 species and collected along the lower reaches of the Dyje River in South Moravia (Czech Republic, central Europe) during 2006-2008: five isolates of Orthobunyavirus Tahyna (TAHV, California group, family Bunyaviridae: three isolations from Aedes vexans (Meigen), one from Ae. sticticus (Meigen), one from Culex modestus Ficalbi); and one isolation of Flavivirus West Nile (WNV, Japanese encephalitis group, family Flaviviridae)-strain Rabensburg (proposed lineage 3 of WNV) from Ae. rossicus (Dolbeshkin et al). All viral isolates were recovered from mosquitoes collected in 2006 (15,882 mosquitoes examined), while no virus was isolated from mosquitoes trapped in 2007 and 2008, when 1,555 and 5,806 mosquitoes were examined, respectively. The population density of local mosquitoes was very low in 2007 and 2008 because of warm and dry summer including a considerably low water table, compared with environmental conditions favorable for mosquito development in 2006. The virus isolation procedure was based on intracerebral inoculation of newborn mice. In parallel, more than one-third of the samples (183 pools consisting of 8,470 individual mosquitoes) were also examined by inoculating Vero cell cultures in Leighton tubes. However, the latter method detected only three of the six virus isolates (including WNV-Rabensburg). Ae. rossicus is a new potential vector for WNV-Rabensburg. This species feeds mostly on mammals including man; this raises the question whether this virus lineage is not adapted to an alternative mosquito-mammal cycle in the South-Moravian natural focus.

Zoonotic mosquito-borne flaviviruses: worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases.

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.

16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens.

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G(+) bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G(+) strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G(-) strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus.

Presence of the mosquito Anopheles hyrcanus in South Moravia, Czech Republic.

During a survey of mosquitoes in the South Moravian lowland area, the mosquito Anopheles hyrcanus (Pallas) (Diptera: Culicidae) was found breeding in an ancient fishpond (Nesyt). It is not clear whether this southern Palaearctic species, a known vector of malaria in Asia which has not been recorded in the Czech Republic until this year, has gone undetected in the past or whether it has recently moved into the region as a result of climate change.

Serological examination of songbirds (Passeriformes) for mosquito-borne viruses Sindbis, Tahyna, and Batai in a south Moravian wetland (Czech Republic).

A total of 178 free-living birds of 14 species of 7 families of Passeriformes sampled in a freshwater reedswamp habitat in southern Moravia in July 2006 were examined for hemagglutination-inhibiting (HI) antibodies to Alphavirus Sindbis (SINV), and bunyaviruses Tahyna (TAHV) and Batai (BATV). Hemagglutination-inhibiting antibody was detected against all three viruses, but at different frequencies: SINV 0.7%, TAHV 14.0%, and BATV 6.8%. The survey indicates circulation of mosquito-borne viruses TAHV and BATV and very low, if any, SINV activity in the area.

[Borrelia burgdorferi sensu lato in ixodid ticks from Ostrava slag heaps]. / Borrelia burgdorferi s.l. v klíst'atech na ostravských haldách.

In 2005 and 2006, Ixodes ricinus ticks were collected on two slag (waste rock) heaps from coal mines in the Ostrava area (North Moravia/Silesia, Czech Republic), Oskar (site A) and Emma (site B), partially covered by vegetation including trees, and at a control forest site near Hlucín (site C). The mean numbers of L. ricinus nymphs and imagoes flagged per person-hour were high: 35.3 nymphs and 12.7 imagoes, at site A, 23.3 and 26.0, respectively, at site B, and 25.4 and 16.8, respectively, at control site C. Using dark-field microscopy, 100 nymphs and 100 imagoes (50 females and 50 males) from each site were examined for borreliae. The mean prevalence rates of Borrelia burgdorferi sensu lato in nymphs and imagoes were 10.0% and 12.0%, respectively, at site A, 10.0% and 24.0%, respectively, at site B, and 13.0% and 17.0%, respectively, at site C. Differences in the prevalence of borreliae in nymphal and adult ticks from the slag heaps and control site were insignificant, but adult ticks from site B compared to site A contained borreliae significantly more frequently. The mean numbers of nymphs and imagoes infected with borreliae flagged per person-hour were 3.3 and 1.2, respectively at site A, 1.5 and 2.9, respectively, at site B, and 3.1 and 2.6, respectively, at site C. Isolation experiments for borreliae were carried out only in 16 ticks containing higher numbers of borreliae, with eight of these being culture-positive. The cultured borreliae were identified by PCR-RFLP as B. garinii (3 isolates: two from site B, one from site C), B. afzelii (4 isolates: one from site A, three from site B) and B. burgdorferi s.s. (one isolate from site A). Surprisingly, the results suggest that slag heaps, when covered by woody vegetation and frequented by humans, could theoretically pose roughly the same LB transmission risk to humans as common forest biotopes.

Serologic survey of the wild boar (Sus scrofa) for Borrelia burgdorferi sensu lato.

Sera of 642 wild boars (Sus scrofa) shot by hunters in ten administrative regions of the Czech Republic during 1995-2000, were tested by indirect hemagglutination assay (IHA) for the presence of anti-Borrelia IgG. Antibodies to Borrelia burgdorferi sensu lato (Bb) were detected in serum samples from all 10 regions, and overall seroprevalence rate was 12.8%. Titres of antibodies ranged from 1:80 to 1:640. Borrelia antibodies were most frequent in the animals from three administrative regions of the Czech Republic: Moravskoslezsky (25.0%), Pardubicky (25.0%) and Královehradecky (24.1%), followed by the regions Plzen sky (16.7%), Olomoucky (13.3%), Jihomoravsky (12.8%), Vysoc ina (11.1%), Jihoc esky (11.1%), Zlínsky (10.3%), and Liberecky (8.9%). Seasonal seroprevalence rate increased in March and April, the peak was in May. The results suggest frequent exposure of wild boars to ixodid ticks infected with Bb, predominantly in rural and forested regions. The study also reviews the importance of wild boar in Lyme borreliosis (LB) ecology. Wild boar serology may provide another means of surveillance of endemic areas of LB.

[Neglected arthropod-borne viral infections in the Czech Republic]. / Opomíjené virové infekce prenásené hematofágními clenovci v Ceské republice.

Central European encephalitis is the most common arthropod-borne virus disease in the Czech Republic, with the mean annual incidence of 6 cases per 100 000 population. However, seven less known arboviruses (Flavivirus West Nile, Bunyavirus Tahyna, Bunyavirus Batai, Bunyavirus Sedlec, Bunyavirus Lednice, Orbivirus Tribee, Uukuvirus Uukuniemi) also circulate in this country, of which West Nile, Tahyna, Tribec and possibly Batai have been reported to cause human disease. Moreover, antibodies against two other pathogenic viruses found in Europe, i.e. Alphavirus Sindbis and Coltivirus Eyach, have been detected in the Czech Republic. The aim of this study is to review briefly the less known arboviruses found in the Czech Republic with emphasis on the taxonomic status, identification of their hosts and vectors, and pathogenicity to humans. These arboviruses can cause febrile illness to aseptic (meningo)encephalitis of unclear etiology. The review points out the possible emergence of these neglected arboviruses in the foreseeable future and provides diagnostic guidance.