Phylogenetic Analysis of Lednice Orthobunyavirus.

Lednice virus (LEDV) has been detected in Culex modestus mosquitoes in several European countries within the last six decades. In this study, phylogenetic analyses of the complete genome segments confirm that LEDV belongs to the Turlock orthobunyavirus (Orthobunyavirus, Peribunyaviridae) species and is closely related to Umbre, Turlock, and Kedah viruses.

Increased Relative Risk of Tick-Borne Encephalitis in Warmer Weather.

Tick-borne encephalitis (TBE) is a serious acute neuroinfection of humans caused by a tick-borne flavivirus. The disease is typically seasonal, linked to the host-seeking activity of Ixodes ricinus (predominantly nymphs), the principal European tick vector species. To address the need for accurate risk predictions of contracting TBE, data on 4,044 TBE cases reported in the Czech Republic during 2001-2006 were compared with questing activity of I. ricinus nymphs monitored weekly at a defined location for the same 6-year period. A time shift of 21 days between infected tick bite and recorded disease onset provided the optimal model for comparing the number of cases of TBE with numbers of questing nymphs. Mean annual distribution of TBE cases and tick counts showed a similar bimodal distribution. Significantly, the ratio of TBE cases to questing nymphs was highest in the summer-autumn period even though the number of questing nymphs peaked in the spring-summer period. However, this pattern changed during a period of extreme meteorological events of flooding and abnormally high temperatures, indicating that changes in climate affect the incidence of TBE. Previous studies failed to link human behavior with changes in incidence of TBE but showed extrinsic temperature impacts arbovirus replication. Hence, we hypothesize the apparent discrepancy between peak nymphal tick activity and greatest risk of contracting TBE is due to the effect of temperature on virus replication in the tick vector. Relative proportions of questing nymphs and the numbers of weeks in which they were found were greater in summer-autumn compared with spring-summer at near-ground temperatures >5°C and at standard day and weekly average temperatures of >15°C. Thus, during the summer-autumn period, the virus dose in infected tick bites is likely greater owing to increased virus replication at higher microclimatic temperatures, consequently increasing the relative risk of contracting TBE per summer-autumn tick bite. The data support the use of weather-based forecasts of tick attack risk (based on daytime ambient temperature) supplemented with weekly average temperature (as a proxy for virus replication) to provide much-needed real-time forecasts of TBE risk.

Abiotic predictors and annual seasonal dynamics of Ixodes ricinus, the major disease vector of Central Europe.

BACKGROUND: Abiotic conditions provide cues that drive tick questing activity. Defining these cues is critical in predicting biting risk, and in forecasting climate change impacts on tick populations. This is particularly important for Ixodes ricinus nymphs, the vector of numerous pathogens affecting humans. METHODS: A 6-year study of the questing activity of I. ricinus was conducted in Central Bohemia, Czech Republic, from 2001 to 2006. Tick numbers were determined by weekly flagging the vegetation in a defined 600 m(2) field site. After capture, ticks were released back to where they were found. Concurrent temperature data and relative humidity were collected in the microhabitat and at a nearby meteorological station. Data were analysed by regression methods. RESULTS: During 208 monitoring visits, a total of 21,623 ticks were recorded. Larvae, nymphs, and adults showed typical bimodal questing activity curves with major spring peaks and minor late summer or autumn peaks (mid-summer for males). Questing activity of nymphs and adults began with ~12 h of daylight and ceased at ~9 h daylight, at limiting temperatures close to freezing (in early spring and late autumn); questing occurred during ~70 % calendar year without cessation in summer. The co-occurrence of larvae and nymphs varied annually, ranging from 31 to 80 % of monitoring visits, and depended on the questing activity of larvae. Near-ground temperature, day length, and relative air humidity were all significant predictors of nymphal activity. For 70 % of records, near-ground temperatures measured in the microhabitat were 4-5 °C lower than those recorded by the nearby meteorological observatory, although they were strongly dependent. Inter-annual differences in seasonal numbers of nymphs reflected extreme weather events. CONCLUSIONS: Weather predictions (particularly for temperature) combined with daylight length, are good predictors of the initiation and cessation of I. ricinus nymph questing activity, and hence of the risk period to humans, in Central Europe. Co-occurrence data for larvae and nymphs support the notion of intrastadial rather than interstadial co-feeding pathogen transmission. Annual questing tick numbers recover quickly from the impact of extreme weather events.

Full-length genome analysis of Calovo strains of Batai orthobunyavirus (Bunyamwera serogroup): implications to taxonomy.

Batai virus (BATV) is a poorly studied arthropod-borne virus belonging to the genus Orthobunyavirus (Bunyamwera serogroup) within the family Bunyaviridae. It has been associated with human influenza-like febrile illness in several Asian, African, and European countries. Calovo virus (CVOV), isolated in 1960 in Slovakia, has been classified as BATV based on high antigenic similarity, and since then both CVOV and BATV were used as synonyms. In order to fully clarify the phylogenetic relationships between CVOV, BATV, and other members of the Bunyamwera serogroup, we performed whole genome sequencing of four CVOV strains isolated in Europe and phylogenetic analyses of all related viruses. The nucleocapsid protein, encoded by the S genomic segment, contains 233 amino acids, 60 of which, putatively critical for protein function, are conserved. Within the CVOV polyprotein encoded by the M genomic segment, putative cleavage sites, N-glycosylation sites, and seven transmembrane regions were identified. The RNA-dependent RNA polymerase, encoded by the L genome segment, exhibits conservation of the three regions known to be conserved among bunyavirus and arenavirus L proteins. Phylogenetic analyses of all three genomic segments of selected orthobunyaviruses clearly revealed that European and Asian/African strains of BATV are phylogenetically different and form two distinct lineages, indicating the existence of two different genotypes of BATV, tentatively named European genotype (with CVOV as a type strain) and Afro-Asian genotype (with BATV as a type strain) of BATV.

Nucleotide variability of Tahyna virus (Bunyaviridae, Orthobunyavirus) small (S) and medium (M) genomic segments in field strains differing in biological properties.

Tahyna virus (TAHV), a mosquito-borne bunyavirus (California group), is frequently associated with inapparent or influenza-like (Valtice fever) infections in humans, rarely leading to atypical pneumonia or meningitis. Field TAHV strains exhibit a high variability in their biological properties with respect to virulence for laboratory mouse, temperature-sensitivity or character of plaques in cell culture. In consideration of the variations in the antigenic properties TAHV and its potential genetic variability, we analyzed complete nucleotide sequences of the small (S) and medium (M) genomic segments of field TAHV strains with different combinations of phenotypic markers. S segment was highly conservative in all analyzed TAHV strains. Within the M segment, the highest variability was observed in the G(C) gene encoding viral envelope protein and to a less extent also in the NSm gene. However, 5' and 3' non-coding regions of M segment, as well as in G(N) gene exhibited highly conservative pattern, indicating its functional importance, but minor or no role in the determination of biological properties of TAHV field strains.

The TICKPRO computer program for predicting Ixodes ricinus host-seeking activity and the warning system published on websites.

The computer program with the acronym TICKPRO (tick prognosis) facilitates medium-range forecasts of the level of host-seeking activity in ticks within a 1-4 day horizon. The program is based on the medium-range weather forecast routinely produced at the Czech Hydrometeorological Institute (CHMI), as well as on previously developed mathematical models describing the correlation of meteorological factors with the host-seeking activity of Ixodes ricinus. These models are based on a 6-year whole-season monitoring of I. ricinus host-seeking activity on experimental fields in the Central Bohemia Region in a typical habitat, oakhornbeam forest, where tick-borne encephalitis (TBE) virus and Borrelia burgdorferi s.str., B. afzelii, and B. garinii have been found. Meteorological data provided by the CHMI meteorological station in Prague-Libus were used, and during the development of the forecasting algorithm micrometeorological data have been collected directly at the tick monitoring sites under selected synoptic weather situations. In the TICKPRO program, the two most successful models utilized ambient air temperature, quantity of atmospheric precipitation, and relative air humidity. The prediction determines 5 levels of risk of attack according to the current proportion of host-seeking ticks, and thus determines the risk of TBE infection. The levels of risk defined by the TICKPRO program are supplemented by instructions on how to prepare oneself for entering sites with potential tick occurrence, how to move around once there, and how to behave on returning home. This warning system is weekly published on websites of National Institute of Public Health and CHMI, Prague, over entire season (March-November).

Integration of a tick-borne encephalitis virus and Borrelia burgdorferi sensu lato into mountain ecosystems, following a shift in the altitudinal limit of distribution of their vector, Ixodes ricinus (Krkonose mountains, Czech Republic).

The altitudinal shift in the limit of Ixodes ricinus occurrence above the previously established altitude of 750 m above sea level has been monitored over the long-term (2002-2008) in the Krkonose Mts. (Giant Mts.), the highest in the Czech Republic, along two vertical transects in their eastern and central parts (600-1020 and 600-1270 m). Ticks were collected by flagging three times annually, and examined individually by PCR or RT-PCR for the presence of Borrelia burgdorferi sensu lato or tick-borne encephalitis virus (TBEV). A total of 5999 I. ricinus ticks were tested. TBEV RNA was detected in 26 ticks at up to 1140 m. Demonstration of TBEV in two larvae of I. ricinus indicates transovarial transmission. Similar infection rates in larvae and nymphs show vertical transmission in TBEV circulation to be very important under these mountain conditions. B. burgdorferi sensu stricto was found at up to 1040-1065 m, Borrelia garinii and Borrelia afzelii up to 1080-1140 m, and Borrelia valaisiana up to 1270 m. The total infection rates of nymphs and larvae were 7.3% and 2%, respectively. B. garinii was the most prevalent (37%), followed by B. afzelii (29%), B. burgdorferi s.s. (11%), and B. valaisiana (9%). Double to quadruple coinfections were detected in 32% of the infected ticks, most frequently B. garinii/B. afzelii. Predominance of B. garinii and B. valaisiana over B. afzelii suggests that small passerine birds moving on the ground are responsible for permanent local populations of I. ricinus in mountain localities with low numbers of small terrestrial mammals. The detection of B. burgdorferi sensu lato and TBEV in host-seeking larvae indicates an autochthonic infection. Upon analysis of the local climate we consider climate warming to be responsible for the spreading of ticks and tick-transmitted pathogens to higher altitudes.

Vertical distribution of the tick Ixodes ricinus and tick-borne pathogens in the northern Moravian mountains correlated with climate warming (Jeseníky Mts., Czech Republic).

A study of the vertical distribution of the common tick Ixodes ricinus and tick-borne pathogens--tick-borne encephalitis virus (TBEV) and genospecies of Borrelia burgdorferi s.l.--was performed in the highest part of the Jeseniky mountain area (the Hrubý Jesenik Mts. with the highest summit Praded, 1,491 m above see level). Altogether 1,253 specimens of all tick stages (607 larvae, 614 nymphs, 8 females and 24 males) were collected at the altitude 990-1,300 m above sea level on 12 collection sites by the flagging method. Altogether 1,207 ticks (8 females, 24 males, 568 nymphs and 607 larvae) were examined for the presence of tick-borne encephalitis virus and B. burgdorferi s.l. None of the samples contained TBEV, 35 samples (6% of adult ticks, 5% of nymphs, 0.7% of larvae) were positive for B. burgdorferi s.l. The most prevalent genospecies were B. afzelii (44%), B. garinii (28%), less frequent were B. burgdorferi sensu stricto (5%), B. valaisiana (3%). The rather large number of ticks (in absolute numbers as well as recounted to the index: average number of nymphs/worker/collection hour) and the presence of all developmental stages clearly demonstrate that there are viable local tick populations in all the sites, and that recorded ticks were not randomly individuals brought into higher altitudes by birds or game animals. The results are compared with the long-term (2002-2007) monitoring of the tick altitudinal distribution in the Krkonose Mts. and the conditions, which allow ticks to establish local populations up to the timberline in both mountain areas, are discussed. Simultaneously, changes in climatic conditions (especially the air temperature) monitored at 3 meteorological stations in the area of the Jeseníky Mts. were compared with the records from another 8 stations in other mountain areas in the Czech Republic. A very similar statistically significant trend of increasing mean air temperatures during the last three decades is found at all analyzed stations. The trend is most pronounced in the spring and summer months with the highest activity of I. ricinus ticks.

Influence of climate warming on tickborne encephalitis expansion to higher altitudes over the last decade (1997-2006) in the Highland Region (Czech Republic).

The steep rise in the incidence of tick-borne encephalitis (TBE) in the 1990s and its subsequent high level in the Czech Republic are not even over the whole territory. It is manifested markedly in the Czech-Moravian Highland region. In the decades of 1971 through 1992, TBE incidence in the Highland Region did not reach the countrywide average. The rise has been noted only since 1997; in the year 2006 TBE incidence in that administrative region was more than double the countrywide average. Analysis of the situation have not found any socio-economic shifts or land-use changes, or in the numbers of game animals, that could have had an effect on TBE incidence. The rise of infections in localities 500 m above sea level (a.s.l.) and more was markedly steeper than that below that altitudinal limit. At those altitudes there has been found an increase in average monthly temperatures exceeding countrywide averages namely in the period of maximum Ixodes ricinus activity (May-August). Detailed analysis of meteorological conditions and comparison with a long-term study of the influence of modifications of the mountain climate in the Krkonose Mts. on I. ricinus tick distribution and the pathogens transmitted by them, have led to the conclusion that likewise in the Czech-Moravian Highland a marked warming had influenced the local population of the vector I. ricinus, caused an activation of foci of TBE, increased contacts of humans with the vector, consequently giving rise to an apparent increase in the incidence of human cases of TBE.

Effects of paclitaxel, docetaxel and their combinations on subcutaneous lymphomas in inbred Sprague-Dawley/Cub rats.

We investigated, whether the effects on paclitaxel, docetaxel or their combinations on T-cell lymphomas in Sprague-Dawley/Cub rats were mainly caused by their different efficiency or combination of different mechanism of action, or limited by metabolic inactivation by P450 enzymes or drug efflux caused by P-glycoprotein (P-gp). Docetaxel most effectively prolonged the survival of rats and the time of lymphoma appearance, inhibited their intravital size and weight after sacrifice. Paclitaxel was poorly effective and combined administration had intermediate effects. Blood levels of both drugs were similar. Repeated administration of paclitaxel, but not docetaxel, decreased its area under concentration, but the effect disappeared 6h after dosing and was not sufficient to explain lower effects of paclitaxel. The faster metabolism of docetaxel than paclitaxel in vitro did not limit its higher efficiency and repeated administration of paclitaxel did not induce its metabolism to decrease its blood levels sufficiently. Likewise, undetectable expression of P-gp protein in tumours could not explain lower effects of paclitaxel, which is a better substrate of P-gp. Docetaxel was three-fold more effective than paclitaxel against P388D1 lymphoma cell line, used as a model of the T-cell lymphoma and combined action was dominated by the effects of docetaxel. Thus, docetaxel was effective against T-cell lymphomas and may be a potential anticancer drug in similar indications.

Extension of Ixodes ricinus ticks and agents of tick-borne diseases to mountain areas in the Czech Republic.

Along with the shift of the hard tick Ixodes ricinus to higher altitudes observed in the Czech Republic a corresponding shift of tick-borne infections to higher altitudes has been expected. Therefore, I. ricinus ticks, mainly nymphs, were investigated for the presence of tick-borne viruses, tick-borne encephalitis (TBE), Tribec and Eyach, and the spirochaete Borrelia burgdorferi sensu lato in the Sumava and Krkonose Mountains (Czech Republic). The TBE virus and different genospecies of B. burgdorferi s.l. were detected by RT-PCR and PCR, respectively. TBE virus was detected in ticks at 620 and 720 m above sea level (a.s.l.), B. burgdorferi s.l. was detected in ticks up to 1065 m a.s.l. Four genospecies of B. burgdorferi s.l. were identified, B. afzelii, B. garinii, B. burgdorferi sensu stricto, and B. valaisiana. Some nymphs carried multiple Borrelia infections. The conditions of tick-borne agents' distribution and potential epidemiological consequences are discussed.

Risk assessment and prediction of Ixodes ricinus tick questing activity and human tick-borne encephalitis infection in space and time in the Czech Republic.

Present risk assessment and prediction of future risk of humans exposed to Ixodes (I.) ricinus tick attacks and, consequently, to tick-borne encephalitis (TBE) virus infection as one of the basic preconditions for successful TBE prevention has been intensively studied in the Czech Republic. An atlas of TBE in the Czech Republic containing predictive maps of I. ricinus high-incidence habitats and TBE risk sites identified by satellite data (Landsat 5 TM with spatial resolution 30 m) at a scale of 1:200,000 over a territory of 52,000 km(2) and maps of human TBE case distribution (1971-2000) has been prepared using remote sensing and geographical information systems technologies. The influence of climate changes on a forest ecosystem inhabited by I. ricinus has been studied in the southern region of the Czech Republic. The analysis of long-term series (1931-2000) of climatologic and phenological characteristics has been carried out. The results are compared with the long-term series of TBE incidence. The influence of weather condition on day-to-day changes of I. ricinus host-seeking activities was studied in 2001-2004. Field observations were realized in the south-eastern periphery of Prague where the experimental plots for tick monitoring were established in a relevant type of forest growth (Querceto-carpinetum). I. ricinus activities were investigated by the flagging method on three plots (200 m(2) each) in weekly intervals (March to November) during 2001-2004. The instruments for micrometeorological observations were installed between the experimental plots. Macrometeorological data were used from the nearby Czech Hydrometeorological Institute first class meteorological observatory. Simple and multiple linear regression and quadratic regression were used to test the relation between weather modification and I. ricinus host-seeking activity. Two preliminary most suitable 'models' are demonstrated.

An attempt to elucidate the increased incidence of tick-borne encephalitis and its spread to higher altitudes in the Czech Republic.

In 1993 the incidence of tick-borne encephalitis (TBE) showed a sharp rise in Central Europe and has remained high since--with some slight fluctuation. This increase is clearly evident in the Czech Republic and it could be roughly characterised as twofold in 1993-2001 in comparison with 1984-1992 (5,240:2,441 human cases). As yet in the Czech Republic the TBE increase has been manifested by: a) higher number of cases in areas well known for TBE occurrence in humans; b) re-emergence in areas where TBE human cases were not observed, or only sporadically, for a long time; c) emergence of TBE in places unknown previously (including highly elevated areas). This phenomenon has not been fully elucidated yet and we would like to contribute to a better understanding of its cause by comparing the present situation with historical data. Besides TBE epidemiological data (1965-2001) we use the twenty-year all season dynamics data of Ixodes ricinus host-seeking activity (1953-1972) supplemented by the data in 1982, 1992 and by meteorological data characterising periods under study. The fluctuation in annual averages of I. ricinus occurrence was irregular. These irregularities when compared with meteorological data can be explained by different meteorological conditions in particular years. In corresponding long-time series, the peaks of I. ricinus occurrence and TBE incidence were reached at the same time. Analyses of relevant meteorological data displayed that joint I. ricinus and TBE maximal occurrence values had been preceded by mild (or warm) autumn seasons allowing a prolongation of I. ricinus activities (including development) up to November at least, thus resulting in a higher tick volume the following year. Based on these data we conclude that the increased TBE incidence rates reported in 1993 and afterwards are attributable to a more abundant occurrence of I. ricinus ticks and that their higher abundance is due to modified climatic conditions in the last decade. Such a situation has appeared in the past as well; however, it was rare and in isolated years only. At that time, although the tick population had been more abundant, in the following year it reached the usual level again. In the 1990s the prolonged mild autumn for consecutive several years led to permanently more abundant populations of I. ricinus. The same conclusion explains the shift of I. ricinus occurrence and of TBE distribution to higher altitudes in Sumava and Krkonose Mountains.