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.

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.

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.

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.

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.

[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.

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.

[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.

[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.

Brucellosis of the common vole (Microtus arvalis).

A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.

Importance of bird-to-bird transmission for the establishment of West Nile virus.

West Nile virus (WNV) is principally considered to be maintained in a mosquito-bird transmission cycle. Under experimental conditions, several other transmission routes have been observed, but the significance of these additional routes in nature is unknown. Here, we derive an expression for the basic reproduction number (R0) for WNV including all putative routes of transmission between birds and mosquitoes to gauge the relative importance of these routes for the establishment of WNV. Parameters were estimated from published experimental results. Sensitivity analysis reveals that R0 is sensitive to transmission between birds via close contact, but not to mosquito-to-mosquito transmission. In seasons or in areas where the mosquito-to-bird ratio is low, bird-to-bird transmission may be crucial in determining whether WNV can establish or not. We explain the use of R0 as a flexible tool to measure the risk of establishment of vector-borne diseases.

European experience with the West Nile virus ecology and epidemiology: could it be relevant for the New World?

A review of West Nile virus (WNV) and the epidemiology of West Nile fever (WNF) in Europe is presented. European epidemics of WNF reveal some general features. They usually burst out with full strength in the first year, but few cases are observed in the consecutive 1 to 2 (exceptionally 3) years, whereas smaller epidemics or clusters of cases only last for one season. The outbreaks are associated with high populations of mosquitoes (especially Culex spp.) caused by flooding and subsequent dry and warm weather, or formation of suitable larval breeding habitats. Urban WNF outbreaks associated with Culex pipiens biotype molestus are dangerous. Natural (exoanthropic, sylvatic) foci of WNV characterized by the wild bird-ornithophilic mosquito cycle probably occur in many wetlands of climatically warm and some temperate parts of Europe; these foci remain silent but could activate under circumstances supporting an enhanced virus circulation due to appropriate abiotic (weather) and biotic (increased populations of vector mosquitoes and susceptible avian hosts) factors. It is very probable that WNV strains are transported between sub-Saharan Africa and Europe by migratory birds. The surveillance system for WNF should consist of four main components: (1) monitoring of mosquito populations and their infection rate; (2) wild vertebrate surveys; (3) sentinel birds (domestic ducks rather than chickens); and (4) monitoring of human disease. In the case of persisting high risk of WNF for humans and equids in certain enzootic areas, immunization against WNF should be considered. For that purpose a commercially available, cross-protective vaccine against Japanese encephalitis could be used.

West Nile virus investigations in South Moravia, Czechland.

Seven virus isolates were obtained from 11,334 mosquitoes after the 1997 Morava River flooding in South Moravia (Czech Republic): 6 strains of Tahyna bunyavirus, California antigenic group (5 from Aedes vexans, 1 from Ae. cinereus), and 1 strain of West Nile flavivirus (WNV) from Culex pipiens. In 1999, one isolate of Tahyna virus from Ae. vexans and one isolate of WNV from Cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there in 1,179 overwintering mosquitoes (mostly Cx. pipiens) in March 2000. The infection rate of mosquitoes with arboviruses was significantly higher in 1997, the year of the flood and an enormously high population density of mosquitoes. Antibodies neutralizing WNV were detected in 13 of 619 (2.1%) hospitalized patients or persons seeking outpatient clinics of the area in 1997. Five of the seroreactors revealed clinical symptoms compatible with West Nile fever: in 2 of them (children), recent infection with WNV was confirmed by a significant increase of antibody titer between acute and convalescent serum samples.

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.

Growth temperature ranges of Borrelia burgdorferi sensu lato strains.

Three strains of Borrelia burgdorferi sensu lato, representing three human pathogenic genomospecies (B31, B. burgdorferi sensu stricto; BR14, B. garinii; BR75, B. afzelii) were grown in BSK-H medium at different temperatures and the spirochaetal cells were counted by dark-field microscopy after 0, 4, 8, 16 and 48 days. Approximate optimum (minimum-maximum) temperatures for the in-vitro growth were found to be 33 degrees C (22-39 degrees C) in strain B31, 35 degrees C (20-40 degrees C) in strain BR75 and 37 degrees C (20-41 degrees C) in strain BR14. Maximum, optimum and minimum growth temperatures seem to be important characteristics of B. burgdorferi s.l. strains, with relevance for the symptomatology, epidemiology and epizoology of Lyme borreliosis.