Tick-borne Encephalitis Virus RNA Research Articles

Molecular detection and phylogenetic analysis of tick-borne encephalitis virus in rodents captured in the transdanubian region of Hungary.

Abstract Tick-borne encephalitis virus (TBEV) infection is a common zoonotic disease affecting humans in Europe and Asia. To determine whether TBEV is present in small mammalian hosts in Hungary, liver samples of wild rodents were tested for TBEV RNA. Over a period of 7 years, a total of 405 rodents were collected at five different geographic locations of the Transdanubian region. TBEV nucleic acid was identified in four rodent species: Apodemus agrarius, A. flavicollis, Microtus arvalis, and Myodes glareolus. Out of the 405 collected rodents, 17 small mammals (4.2%) were positive for TBEV. The present study provides molecular evidence and sequence data of TBEV from rodents in Hungary.

Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment, Poland.

Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is an arthropod-borne virus, an etiologic agent of tick-borne encephalitis (TBE), a human infection involving the central nervous system. The disease is endemic in a large region in Eurasia, where it is transmitted mainly by Ixodes ricinus and Ixodes persulcatus ticks. It is known that also Dermacentor reticulatus is involved in circulation of TBEV. However, the current knowledge of D. reticulatus importance in TBE epidemiology is still insufficient. A total of 471 adult D. reticulatus ticks were collected by flagging vegetation in the Białowieża Primeval Forest, Biebrza National Park, Masurian Landscape Park (North-Eastern Poland) and in the city of Warsaw in the years 2007–2010. All collected ticks were examined individually for the presence of RNA of TBEV using nested RT-PCR assay. Positive results were noted in all investigated localities with the infection rate ranging from 0.99 to 12.5 % with a total mean of 2.12 %. The difference in the percentage of infective males and females was not statistically significant.

The Stress Granule Component TIA-1 Binds Tick-Borne Encephalitis Virus RNA and Is Recruited to Perinuclear Sites of Viral Replication To Inhibit Viral Translation

Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA-binding proteins involved in the repression of initiation of translation of cellular mRNAs and in the formation of stress granules. We show that TIA-1 and TIAR interact with viral RNA in TBEV-infected cells. During TBEV infection, cytoplasmic TIA-1 and TIAR are recruited at sites of viral replication with concomitant depletion from stress granules. This effect is specific, since G3BP1, another component of these cytoplasmic structures, remains localized to stress granules. Moreover, heat shock induction of stress granules containing TIA-1, but not G3BP1, is inhibited in TBEV-infected cells. Infection of cells depleted of TIA-1 or TIAR by small interfering RNA (siRNA) or TIA-1(-/-) mouse fibroblasts, leads to a significant increase in TBEV extracellular infectivity. Interestingly, TIAR(-/-) fibroblasts show the opposite effect on TBEV infection, and this phenotype appears to be related to an excess of TIA-1 in these cells. Taking advantage of a TBE-luciferase replicon system, we also observed increased luciferase activity in TIA-1(-/-) mouse fibroblasts at early time points, consistent with TIA-1-mediated inhibition at the level of the first round of viral translation. These results indicate that, in response to TBEV infection, TIA-1 is recruited to sites of virus replication to bind TBEV RNA and modulate viral translation independently of stress granule (SG) formation. This study (i) extends previous work that showed TIA-1/TIAR recruitment at sites of flavivirus replication, (ii) demonstrates that TIAR behaves like TIA-1 as an inhibitor of viral replication using an RNA interference (RNAi) approach in human cells that contradicts the previous hypothesis based on mouse embryonic fibroblast (MEF) knockouts only, (iii) demonstrates that tick-borne encephalitis virus (TBEV) is capable of inducing bona fide G3BP1/eIF3/eIF4B-positive stress granules, (iv) demonstrates a differential phenotype of stress response proteins following viral infection, and (v) implicates TIA-1 in viral translation and as a modulator of TBEV replication.

Surveillance of tick-borne encephalitis virus in wild birds and ticks in Tomsk city and its suburbs (Western Siberia)

To study the role of wild birds in the transmission of tick borne encephalitis virus (TBEV), we investigated randomly captured wild birds bearing ixodid ticks in a very highly endemic TBE region located in Tomsk city and its suburbs in the south of Western Siberia, Russia. The 779 wild birds representing 60 species were captured carrying a total of 841 ticks, Ixodes pavlovskyi Pom., 1946 (n=531), Ixodes persulcatus P. Sch., 1930 (n=244), and Ixodes plumbeus Leach. 1815 (n=66). The highest average number of ticks per bird in a particular species was found for the fieldfare (Turdus pilaris Linnaeus, 1758) (5.60 ticks/bird) and the tree pipit (Anthus trivialis Linnaeus, 1758) (13.25 ticks/bird). Samples from wild birds and ticks collected in highly endemic periods from 2006 to 2011 were tested for the TBEV markers using monoclonal modified enzyme immunoassay (EIA) and RT-PCR. TBEV RNA and antigen were found in 9.7% and 22.8% samples collected from wild birds, respectively. TBEV markers were also detected in 14.1% I. persulcatus ticks, 5.2% I. pavlovskyi, and 4.2% I. plumbeus ticks collected from wild birds. Two TBEV strains were also isolated on PKE (pig kidney embryo) cells from fieldfare and Blyth's reed warbler (Acrocephalus dumetorum Blyth, 1849). Sequencing of 5′-NCR of TBEV revealed that all TBEV isolates belong to Far Eastern (dominate) and Siberian genotypes. Several phylogenetic subgroups included TBEV sequences novel for the Tomsk region. Our data suggest that wild birds are potential disseminators of TBEV, TBEV-infected ixodid ticks, and possibly other tick-borne infections.

Tick-borne Encephalitis Virus, Zealand, Denmark, 2011

Tick-borne Encephalitis Virus, Zealand, Denmark, 2011

Tick-borne Encephalitis Associated with Consumption of Raw Goat Milk, Slovenia, 2012

Tick-borne encephalitis (TBE) developed in 3 persons in Slovenia who drank raw milk; a fourth person, who had been vaccinated against TBE, remained healthy. TBE virus RNA was detected in serum and milk of the source goat. Persons in TBE-endemic areas should be encouraged to drink only boiled/pasteurized milk and to be vaccinated.

Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA

Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.

Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

BackgroundTick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.MethodsPrimers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.ResultsThe sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.ConclusionTBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.

Prevalence of Tick-Borne Encephalitis Virus in Ixodid Ticks Collected from the Republic of Korea During 2011–2012

ObjectivesIn this study, we investigated the prevalence of tick-borne encephalitis virus (TBEV) in ixodid ticks from various regions of the Republic of Korea (ROK) during 2011–2012 to identify whether TBEV is circulating and to determine the endemic regions of TBEV. MethodsWe examined for the presence of RNA of TBEV by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) using ixodid ticks captured in 25 localities of 10 provinces. Ticks were collected by the flagging and dragging method or using sentinel BG traps at forests, grass thickets, and grassland. A total of 13,053 ticks belonging to two genera and four species were collected and pooled (1292 pools), according to collection site, species of tick, and developmental stage. ResultsAmong 1292 pools, the envelope (E) protein gene of TBEV was detected using RT-nested PCR in 10 pools (3 pools of the 1,331 adult ticks and 7 pools of the 11,169 nymph ticks) collected from Gangwon-do province, Jeonrabuk-do province, and Jeju Island. The minimum infection rates for TBEV of Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis were 0.06%, 0.17%, and 2.38%, respectively. Phylogenetic analysis based on the partial E protein gene was performed to identify relationships between the TBEV strains. This showed that 10 Korean strains clustered with the Western subtype. ConclusionIn this study, we demonstrated that TBEV-infected ticks have been distributed in the ROK, combined with our previous results. These results suggest that TBEV may exist in the ROK, and H. longicornis, H. flava, and I. nipponensis may be potential vectors of TBEV. In addition, these results emphasize the need for further epidemiological research of TBEV.

The correlation between tick (Ixodes persulcatus Sch.) questing behaviour and synganglion neuronal responses to odours

We examined the behavioural and electrophysiological responses of taiga ticks (Ixodes persulcatus) to several olfactory stimuli: Osmopherone® (5-a-androst-16-en-3-ol), Osmopherine® (butanoic and 3-methylbutanoic acids), DEET® (N,N-diethyl-meta-toluamide), ethanol (96%), and water (control stimulus). To study individual tick behavioural reactions to these stimuli, we used a Y-shaped glass maze (n=50). To study the electrophysiological reactions of the ticks’ synganglia to these olfactory stimuli, we recorded the shifts of total potential (TP) of pre-oesophageal neurons in response to odour stimulation of Haller’s organ (n=25). We found that Osmopherine® attracted ticks and frequently evoked negative shifts of TP, whereas the response to Osmopherone® did not differ from the reaction to water. DEET® and ethanol acted as tick repellents and generally evoked positive shifts of TP. We also tested each tick for its questing height (QH) on a glass rod that was at an incline of 75°, and we tested for the presence of pathogens i.e., DNA of Borrelia burgdorferi sp. s.l. and RNA of tick-borne encephalitis virus (TBEV). The degree of response to Osmopherine® positively correlated with the QH. The ticks with the highest values of QH showed a greater prevalence of the tick-borne pathogen Borrelia sp. s.l. compared with the ticks that did not reach the maximum QH. The present results show a correlation between the electrophysiological reaction of the synganglia of ticks and their behavioural responses to different odours.

Patterns of Tick-Borne Encephalitis Virus Infection in Rodents in Slovenia

Tick-borne encephalitis virus (TBEV) is the most important causative agent of arboviral infection in Europe, causing neurologic symptoms. The incidence of the disease has greatly increased over the past decades, and in the meantime, some changes in spatial distribution of TBE cases have been observed. Therefore, it is important to recognize the distribution of endemic areas, to use preventive measures successfully. In this study, rodents from all over Slovenia were evaluated as suitable sentinels for TBEV distribution. Rodents from four species (Myodes glareolus, Apodemus flavicollis, Apodemus sylvaticus, and Apodemus agrarius) were screened for the presence of TBEV antibodies with immunofluorescence assay; the antibodies were detected in 5.9% of sera. The prevalence of infection varied according to the rodent species and according to the region of trapping. Select rodents were also screened for the presence of TBEV RNA in several organs. Both analyses showed higher rate of infection in bank voles, which also produced higher titers of anti-TBEV antibodies and a higher TBEV RNA viral load compared with mice. The regional prevalence of infection in rodents can be correlated with the incidence of disease. Molecular results indicate that the virus can be detected in the organs of the rodents for longer periods, indicating prolonged infections of the rodent hosts by the virus. Rodents can therefore be used as a useful indicator of the circulation of TBEV in an area.

Acute myositis associated with the initial phase of tick-borne encephalitis

We present a case of clinically overt acute myositis associated with the initial phase of tick-borne encephalitis in a ten-year-old boy. The diagnosis of the disease was confirmed by detection of tick-borne encephalitis virus RNA in blood prior to the development of serum specific antibodies and the second phase of the disease.

Quantitations of Borrelia and Bartonella DNA and tick-borne encephalitis virus RNA in Ixodes persulcatus ticks collected in Chelyabinsk region

Borrelia and Bartonella DNA and tick-borne encephalitis virus (TBEV) RNA were detected in Ixodes persulcatus Schulze ticks collected in May 2009 in Chelyabinsk region, Ural, Russia by means of PCR with subsequent electrophoresis or real-time PCR with fluorescent hydrolysis probes. Borrelia garinii and Borrelia afzelii DNA were found in 36.6 ± 5.0% of taiga ticks with bacterial loads 101–106 DNA copies per an infected tick. Bartonella quintana and Bartonella henselae DNA were revealed in 38.9 ± 5.2% of I. persulcatus with prevalence of samples with high bacterial loads of more than 1000 genome-equivalents. The tick-borne encephalitis virus RNA of Siberian genetic subtype was detected in 16.9 ± 4.3% of I. persulcatus by using reverse transcription with subsequent real time PCR with fluorescent hydrolysis probe. Quantitation of viral loads varied in a range 104–107 genome-equivalents per a tick exceeded the amounts of tick-borne bacteria. Experimentally observed frequencies of mixed infections with Borrelia and Bartonella in 13.0% of ticks fitted well to theoretical estimations based on independent distribution of the tick-borne bacteria among vectors.

European Subtype Tick-borne Encephalitis Virus inIxodes persulcatusTicks

European Subtype Tick-borne Encephalitis Virus in<i>Ixodes persulcatus</i>Ticks

Prevalence of tick-borne encephalitis virus in Ixodes ricinus and Dermacentor reticulatus ticks collected from the Lublin region (eastern Poland)

A total of 875 nymphal and adult Ixodes ricinus ticks and 148 adult Dermacentor reticulatus ticks were collected by flagging lower vegetation in the Lublin region (eastern Poland) and examined for the presence of RNA of tick-borne encephalitis virus (TBEV) by nested RT-PCR. The minimum infection rate of I. ricinus ticks with TBEV amounted to 1.6% while the infection rate of D. reticulatus ticks was 10.8%. The results suggest that D. reticulatus may be a potential vector of TBEV in Central Europe.

Zoological prerequisites of human tick-borne infections in the Northern Urals

The results of studies in the pessimum of the taiga tick (Ixodes persulcatus Sch., 1930) range in the Urals have shown that this species occurs in the regions at latitudes of up to 60°50′ N. In Northern Ural mountains, the ticks show preference for secondary small-leaved forests growing on southern slopes, where their abundance along census routes reaches 7 ind./km. The abundance and distribution of taiga ticks hosts, birds and small mammals, have been estimated in the main types of their habitats. Larvae and nymphs of the taiga tick have been found on nine bird and seven small mammal species, with the main parasitic load falling on the northern red-backed vole (Clethrionomys rutilus P., 1779), which is widespread and abundant in the greater part of the study area. Molecular biological analysis of I. persulcatus ticks and blood samples from their hosts has revealed the presence of nucleic acids of pathogenic microorganisms A. phagocytophilum, E. muris, and A. burgdorferi sensu lato, the RNA of tick-borne encephalitis virus, and the DNA of Babesia microti.

Tick-borne encephalitis virus in ticks in Finland, Russian Karelia and Buryatia

Tick-borne encephalitis (TBE) is a central nervous system infection caused by a flavivirus [tick-borne encephalitis virus (TBEV)], transmitted by Ixodes ticks and endemic in a large region in Eurasia. We collected 2411 ticks from Finland and Russia in 2003-2008, screened them for TBEV by RT-PCR and isolated and analysed eight strains belonging to all three TBEV subtypes; in addition, we obtained two European-subtype strains from human serum samples. TBEV RNA prevalence in unengorged ticks was approximately 1 % both in the northernmost TBE-endemic areas of Europe in Finland and Russian Karelia, and in Siberia in Buryatia. In Finland, both Ixodes ricinus and Ixodes persulcatus ticks were found from distinct areas and, in Russian Karelia, were overlapping in the same study site. TBEV E and NS3 gene sequences obtained showed a variability of 0-4 % within European-subtype strains, 2-9 % for Siberian-subtype strains and 3-13 % for Far Eastern-subtype strains.

Integration of a Tick-Borne Encephalitis Virus andBorrelia burgdorferisensu lato into Mountain Ecosystems, Following a Shift in the Altitudinal Limit of Distribution of Their Vector,Ixodes ricinus(Krkonoše 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.

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany

Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a former risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from volunteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized.

Vertical transmission of tick-borne encephalitis virus between generations of adapted reservoir small rodents

Vertical transmission of tick-borne encephalitis virus (TBEV) between generations of the small rodents—red voles Myodes rutilus Pallas (previously known as Clethrionomys rutilus Pallas) was shown for naturally infected reservoir hosts and after experimental infection with different sublethal doses of the viral strains. For wild red voles and for their progeny born in 240–280 days after experimental infection of their parents the TBEV was detected in up to 90% of samples by RT-PCR, ELISA and bioassays. Small amounts of the TBEV RNA found in embryos, placenta and blood cells could serve as evidence of prenatal transmission. Postnatal transfer of the virus might occur through the rodent milk. Analysis of the TBEV E gene nucleotide sequences of RT-PCR products revealed missense mutations resulting in amino acid substitution K280Q in newborn red vole in comparison with its parent female. Taken together, the data confirmed the TBEV vertical transmission among generations of its adapted mammal reservoir hosts. The virus transfer might occur before, during and/or after birth of the small rodents with high frequencies. In the wild it could provide the TBEV long-term persistence in mammal hosts without an (any) involvement of arthropod vectors thus selecting dangerous mammal-adapted variants.