Detection Of Tick-borne Encephalitis Virus Research Articles

Detection of tick-borne encephalitis virus in ear tissue and dried blood spots from naturally infected wild rodents

BackgroundTick-borne encephalitis virus (TBEV) can cause severe neurological disease in humans. Its geographical distribution is expanding in Western Europe with unresolved causes and spatial patterns, necessitating enhanced surveillance. Monitoring the virus in the environment is complicated, as it usually relies on destructive sampling of small rodents to test organs for TBEV, which in addition to ethical considerations also raises issues for long-term monitoring or longitudinal studies. Moreover, even when the virus is not detected in the blood or organs of the rodent, TBEV can still be transmitted from an infected tick to uninfected ticks feeding nearby. This is due to the ability of TBEV to replicate and migrate locally within the epidermis of small mammals, including those that do not appear to have systemic infection. This suggests that the virus may be detectable in skin biopsies, which has been confirmed in experimentally infected laboratory rodents, but it remains unknown if this sample type may be a viable alternative to destructively obtained samples in the monitoring of natural TBEV infection. Here we test ear tissue and dried blood spot (DBS) samples from rodents to determine whether TBEV-RNA can be detected in biological samples obtained non-destructively.MethodsRodents were live-trapped and sampled at three woodland areas in The Netherlands where presence of TBEV has previously been recorded. Ear tissue (n = 79) and DBSs (n = 112) were collected from a total of 117 individuals and were tested for TBEV-RNA by real-time RT-PCR.ResultsTBEV-RNA was detected in five rodents (4.3% of tested individuals), all of which had a TBEV-positive ear sample, while only two out of four of these individuals (for which a DBS was available) had a positive DBS. This equated to 6.3% of ear samples and 1.8% of DBSs testing positive for TBEV-RNA.ConclusionsWe provide the first evidence to our knowledge that TBEV-RNA can be detected in samples obtained non-destructively from naturally infected wild rodents, providing a viable sampling alternative suitable for longitudinal surveillance of the virus.Graphical

Transmissible tick-borne infections in the south of the Far East

Natural foci of tick-borne infections associated with tick-borne encephalitis virus (TBEV), borrelia (Borrelia burgdorferi sensu lato), including Borrelia miyamotoi, anaplasma, ehrlichia and rickettsia are widespread in the Primorsky Krai. The carriers of these pathogens are ixodid ticks. The population of Primorsky Krai meets with ticks in natural biotopes, as well as in anthropurgic foci. The aim of the study is to give a comparative assessment of the epizootic activity in the natural foci of transmissible tick-borne infections in the south of Primorsky Krai in the epidemic seasons of 2017–2020. In this periodixodid ticks (3778 samples), taken from humans in natural foci in the Primorsky Territory, were studied. The TBEV antigen was determined by enzyme-linked immunosorbent assay (ELISA), genetic markers of pathogens were detected by real-time polymerase chain reaction (RT-PCR). The incidence of tick-borne infections has been analyzed. Based on the results of monitoring the infection of ixodid ticks, we found that 26.9 % of the studied samples were infected with various pathogens of tick-borne infections. Low infection rate of TBEV in ixodid ticks and high infection with pathogens of a bacterial nature were established. TBEV antigen was detected in 30 cases (1.4 %), TBEV RNA was detected in 20 cases (0.7 %), Borrelia burgdorferis. l. DNA — in 820 cases (30.7 %), ehrlichia — in 64 (2.4 %), anaplasma — in 55 (2.1 %).Genetic marker of Rickettsia spp. was detected in 3 cases (0.9 %) out of 322 examined ixodid ticks, B. miyamotoi — in 26 cases (6.9 %) out of 373 examined samples. A positive correlation was noted between the incidence rates and the cases of detection of TBEV and Lyme borreliosis in ticks. The results obtained indicate the need for annual epidemiological monitoring of infections transmitted by ixodid ticks, to determine the real epidemic situation and the activity of the functioning natural foci transmissible tick-borne infections on the territory of Primorsky Krai.

Tick burden on European roe deer (Capreolus capreolus) from Saxony, Germany, and detection of tick-borne encephalitis virus in attached ticks.

Southern Germany is known as tick-borne encephalitis (TBE) risk area; however, the north of the country is almost free of human TBE cases. Due to its location in the transition zone between TBE risk areas and areas with only sporadic cases, Saxony is of importance in the surveillance of TBE. Roe deer (Capreolus capreolus), showing high seroprevalence of TBE virus (TBEV) antibodies, are considered to be sentinels for TBE risk assessment. Thus, roe deer could be used as indicators helping to better understand the focality of the TBEV in nature and as a possible source to isolate TBEV. Therefore, the aims of this study were to examine roe deer coats for the presence of ticks to establish the tick burden and to detect the TBEV in attached ticks. One hundred thirty-four roe deer coats were provided by hunters from the Hunting Association in Saxony (August 2017-January 2019). The coats were frozen at - 80°C and after de-freezing examined on both sides-inside and outside. Attached and nonattached ticks were collected, morphologically identified and tested using real-time RT-PCR for the presence of TBEV. In total, 1279 ticks were found on 48 coats. The predominant species was Ixodes ricinus (99.76%; n = 1276). Three remaining specimens were Ixodes spp. (0.16%, 1 female and 1 nymph) and Dermacentor reticulatus (0.08%, 1 male). The average infestation rate was 26.7 (SD = 69.5), with maximum of 439 ticks per animal. Females were the dominant life stage of ticks (n = 536; 42%), followed by nymphs (n = 397; n = 31.1%), males (n = 175; 13.7%), and larvae (n = 168; 13.2%). Only half of collected ticks were attached (n = 662; 51.8%). TBEV was detected only in one tick out of 1279 tested ticks. It was a female infesting a roe deer from Saxon Switzerland-East Ore Mountain. The results show that the method used in this study is not sufficient as a sentinel marker to predict TBEV spreading in nature. Although previous studies demonstrated the usefulness of serological testing of roe deer in order to trace TBE-endemic regions, using ticks attached to them to get virus isolates is not productive.

First detection of tick-borne encephalitis virus in Ixodes ricinus ticks and their rodent hosts in Moscow, Russia

Here, we report the first confirmed autochthonous tick-borne encephalitis case diagnosed in Moscow in 2016 and describe the detection of tick-borne encephalitis virus (TBEV) in ticks and small mammals in a Moscow park.The paper includes data from two patients who were bitten by TBEV-infected ticks in Moscow city; one of these cases led to the development of the meningeal form of TBE. Both TBEV-infected ticks attacked patients in the same area. We collected ticks and trapped small mammals in this area in 2017. All samples were screened for the presence of pathogens causing tick-borne diseases by PCR. The TBEV-positive ticks and small mammals’ tissue samples were subjected to virus isolation. The sequencing of the complete polyprotein gene of the positive samples was performed.A total of 227 questing ticks were collected. TBEV was detected in five specimens of Ixodes ricinus. We trapped 44 small mammals, mainly bank voles (Myodes glareolus) and pygmy field mice (Apodemus uralensis). Two samples of brain tissue from bank voles yielded a positive signal in RT-PCR for TBEV. We obtained six virus isolates from the ticks and brain tissue of a bank vole. Complete genome sequencing showed that the obtained isolates belong to the European subtype and have low diversity with sequence identities as high as 99.9%. GPS tracking showed that the maximum distance between the exact locations where the TBEV-positive ticks were collected was 185 m. We assume that the forest park had been free of TBEV and that the virus was recently introduced.

First detection of TBE virus in ticks and sero-reactivity in goats in a non-endemic region in the southern part of Switzerland (Canton of Ticino)

In Switzerland, tick-borne encephalitis (TBE) is a notifiable human disease with an average of 210 cases per year in the last 10 years (2008–2017). A national surveillance conducted in 2009 reported a prevalence of 0.46% for tick-borne encephalitis virus (TBEV) detected in ticks, which is in accordance with the prevalences found in Europe from 0.1%–5%. The Canton of Ticino in the southern part of Switzerland, geographically separated from the rest of the national territory by the Alps, is considered a non-endemic region, as no autochthonous clinical cases and no TBEV presence in ticks have ever been reported. In order to understand the epidemiological situation in Ticino, we conducted a large study investigating the TBEV presence in field-collected Ixodes ricinus ticks and in goat and human sera. Goats and sheep were considered as sentinel hosts showing persistence of antibodies also after 28 months in the absence of symptoms; this longevity supports the data validity to characterize an area with the TBEV status. The goat sera collection was composed of a total of 662 samples from 37 flocks. The total seroprevalence was 14.6%. 39 (40%) of the 97 SNT-positive samples showed an antibody titer ≥ 1:120 which indicates recent infection and consequently the probable presence of active foci among the pastures frequented by the goats belonging to 10 flocks. In total, 51 owners participated in the study and all were TBEV antibody-free.A total of 12’052 I. ricinus ticks (nymphs and adults) were collected and 1’371 pools were tested using quantitative real-time RT-PCR. Only one positive pool was reported with a prevalence of 0.35%. Metagenomic analysis revealed that the TBEV strain isolated from the ticks collected in Ticino is closely related to 2 strains coming from the Canton of Valais (99.1% and 98.7% identity, respectively), a neighbouring region of the Canton of Ticino. These two Cantons are close together but separated by high mountains (Alps) and we hypothesize that infected ticks were transported by wild animals from Valais into the Valle Maggia in Ticino where we found positive ticks. In conclusion, our data show for the first time the presence of TBEV in ticks and the related sero-reactivity in goats, confirming the presence of TBEV in the environment of the Canton of Ticino. Further surveillance studies will have to be conducted to follow the persistence of TBEV in this region.

Tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes ricinus ticks collected from recreational islands in southern Norway

The aim of this study was to determine the occurrence of tick-borne pathogens of medical importance in questing ticks collected from five recreationally used islands along the Norwegian coastline. Furthermore, since coinfection may affect the disease severity, this study aimed to determine the extent of coinfection in individual ticks or co-localization of tick-borne pathogens. In all, 4158 questing Ixodes ricinus ticks were analyzed. For detection of tick-borne encephalitis virus (TBEV), nymphs (3690) were analyzed in pools of ten. To detect Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis, 468 nymphs were analyzed individually. A total of five nymph pools was infected with TBEV, giving an overall prevalence of 0.14%. In the individually analyzed ticks, B. burgdorferi s. l. (15.6%), Candidatus N. mikurensis (11%), A. phagocytophilum (1.4%) and B. miyamotoi (0.9%) were detected. Coinfection was found in 3.3% of the ticks, and the only dual infection observed was with B. afzelii and Candidatus N. mikurensis. This association was significantly higher than what would occur by random chance.

Bioluminescent detection of tick-borne encephalitis virus in native ticks

Easy-to-use and fast bioluminescent immunoassay for tick-borne encephalitis virus in natural ticks based on the hybrid protein 14D5a-Rm7 was developed. The approach holds much promise with a view of practical applicability.

ПРИМЕНЕНИЕ МЕТОДА ПЦР В РЕАЛЬНОМ ВРЕМЕНИ ДЛЯ ДИАГНОСТИКИ ИНФЕКЦИЙ, ПЕРЕДАЮЩИХСЯ ИКСОДОВЫМИ КЛЕЩАМИ

The urgent detection of tick-borne pathogens in attached ticks or in human blood followed by the prophylaxis of infection with specific immunoglobulin and/or antibiotics is one of the most reliable measures to prevent human disease. The aim of this work was to evaluate the usability of real-time PCR (RT-PCR) assay in early detection of tick-borne infections. Using commercial RT-PCR assay (Amplisens, Russia) we studied 361 Ixodes persulcatus ticks, 33 Dermacentorsp. ticks and 448samples of human blood delivered by people attacked by ticks in Irkutsk region during 2013–2014 for presence of DNA/RNA of tick-borne encephalitis virus (TBEV), Borrelia burgdorferi sensu lato (B.b.s.l.), Anaplsma phagocytophilum and Ehrlichia chaffeensis/E.muris. The ELISA assay and direct microscopy were used to validate the detection of TBEV and B.b.s.l. respectively. The highest prevalence of tick-borne infections was observed for I.persulcatus ticks – 6,9; 23,8; 6,9; and 11,9% for TBEV, B.b.s.l., A.phagocytophillum and E.chaffeensis/E.muris respectively. The Dermacentorsp. ticks were less frequently infected with abovementioned pathogens – 3,0; 6,1; 3,0 and 9,1% respectively. The prevalence of infection in blood samples was lowest and comprised 2,5; 4,2; 2,2 and 4,5% respectively. Mixed infections were documented in 6,5% of ticks and 0,7% of human blood samples. The testing of blood samples in ELISA and testing of ticks in ELISA and direct microscopy corresponded to the results, obtained by RT-PCR. Thus, RT-PCR is valuable and reliable approach for urgent detection and prophylaxis of tick-borne infections.

Detection of tick-borne encephalitis virus in I. ricinus ticks collected from autumn migratory birds in Latvia

Birds have a potential of spreading ticks via bird migration routes. In this study, we screened 170 ticks removed during autumn 2010 from 55 birds belonging to 10 species for the presence of tick-borne encephalitis virus (TBEV). In total, TBEV RNA was detected in 14% of I. ricinus tick samples obtained from different birds species. The results of this study indicate the possible role of migrating birds in the dispersal of TBEV-infected ticks along the southward migration route.

Serological Evidence of Tick-Borne Encephalitis Virus Infection in Rodents Captured at Four Sites in Switzerland

In a previous study, the presence of tick-borne encephalitis virus (TBEV) in questing Ixodes ricinus L. ticks and in field derived ticks that engorged on small mammals (n = 9,986) was investigated at four sites located in a TBE area in Switzerland. Two of these sites were already recognized as TBE foci (Thun and Belp) and the screening of ticks revealed the presence of TBEV in ticks at a third site, Kiesen, but not at the fourth one, Trimstein. The aim here was to test another approach to detect TBE endemic areas. Sera from 333 small mammals (Apodemus flavicollis, A. sylvaticus, Myodes glareolus) captured in 2006 and 2007 at the four sites were examined for the presence of antibodies against TBEV using immunofluorescence and avidity tests. Overall the prevalence of antibodies against TBEV in rodents reached 3.6% (12/333). At two sites known as TBE foci, Thun and Belp, anti-TBEV antibodies were detected in 9.9% (9/91) and 1.6% (1/63) of rodent sera, respectively. At the third site, Kiesen, recently identified as a TBE focus by the detection of TBEV in ticks, anti-TBEV antibodies were detected in 1.8% (2/113) of rodent sera. Finally, at Trimstein, none of the examined rodent sera had antibodies against TBEV (0/66). This study shows another approach to detect TBE foci by testing antibodies in small mammal sera that is less time-consuming and less expensive than molecular tools.

Häufigkeit des Frühsommer-Meningoenzephalitis-Virus und von Rickettsien in Zecken aus dem Burgenland (Österreich)

Large areas of the Central European country Austria are known to be endemic for tick-borne encephalitis virus (TBEV). However, so far only limited data are available for the prevalence, distribution and species composition of rickettsiae of the spotted fever group in Austria. In the present study 306 ticks were collected in summer 2007. Location of sampling was Neudörfl, a known TBEV endemic area in the district of Burgenland in Eastern Austria. 286 ticks belonged to the species Ixodes ricinus and 20 ticks were identified as Dermacentor spp. Detection of TBEV and rickettsial species was done by polymerase chain reaction. We were not able to detect TBEV in any of the ticks examined. However 16 ticks, exclusively of the species Ixodes ricinus showed positive results for rickettsiae. 14 out of 16 rickettsia-positive ticks contains sufficient DNA for a sequence determination. The sequencing of the citrate synthase genes resulted exclusively in the identification of Rickettsia helvetica. The results show that TBEV was not at all or in a low frequency circulating in a known endemic area in summer 2007. However, for the first time the prevalence of 5.7% of Rickettsia helvetica was determined in a tick population in Eastern Austria.

Quality control assessment for the PCR diagnosis of tick-borne encephalitis virus infections

Background Reverse transcriptase-polymerase chain reaction (RT-PCR) is an efficient method for the early detection of tick-borne encephalitis virus (TBEV) RNA in blood and serum samples taken prior to the appearance of antibodies. Improved diagnostics are critical for optimally detecting and managing TBE infections and quality control measures are therefore essential. Objective To assess the diagnostic quality of laboratories by performing an external quality assurance (EQA) programme for the molecular detection of TBE infections. Study design A panel of 12 prepared human plasma samples were distributed and tested for the presence of TBEV-specific RNA. The panel comprised eight samples spiked with different TBEV strains of European, Siberian and Far Eastern subtypes, and included a 10-fold dilution series. Two specificity controls consisted of a sample with Louping ill virus (LIV) and a sample with a pool of four other flaviviruses, and two negative control samples were further included. Results Twenty-three laboratories from 16 European and 2 non-European countries participated in this EQA programme. Only two participants correctly identified all samples. Nine laboratories correctly identified 75.0–91.7% of the samples; seven laboratories correctly identified 54.5–66.7% and five laboratories correctly identified ≤50%. Conclusions The EQA programme provides information on the quality of the RT-PCR methods used by the participating laboratories and indicates that most of these need to improve sensitivity and specificity of their molecular assays for TBEV.

A nested RT-PCR for the detection of Tick-borne Encephalitis Virus (TBEV) in ticks in natural foci

We have developed a sensitive nested reverse-transcriptase polymerase chain reaction assay (n RT-PCR) for the detection of the tick-borne encephalitis virus (TBEV) RNA, especially in ticks. The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences as well as by the sequencing of the first and second amplificate, by Southern blot hybridization with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive. The detection limit is about 20 fg of TBEV RNA per PCR run (25 microliters), or a single positive tick, i.e. (adult or nymph). The method can be used for comparative studies of the epidemiological situation, as well as for the screening of natural foci for the presence and circulation of TBEV or for the detection of TBEV-genome-sequences in clinical materials.

Detection of tick-borne encephalitis virus by sample transfer, plaque assay and strand-specific reverse transcriptase polymerase chain reaction: what do we detect?

Experimental inoculation of mice provides a well characterized model for studying infection with tick-borne encephalitis virus (TBEV), a flavivirus pathogenic for humans. Conflicting data on the kinetics of viremia and the development of virus titers in the brain, however, were only recently shown to have resulted from the use of assay systems with different levels of sensitivity in the titration of TBEV i.e. plaque assay or sample transfer into naive recipient mice. Theoretically, RT-PCR could extend further the detectability to antibody-neutralized virus and when undertaken strand-specifically discriminate active replication from the mere presence of TBEV. We have compared the conventional methods for detection of TBEV with a newly devised RT-PCR method. As expected, RT-PCR, in contrast to the infectivity assays, detected antibody-neutralized virus. Furthermore, the mere presence or active replication of the virus could be differentiated by strand-specific RT-PCR. Plaque assay and sample transfer, in contrast, both detected only infectious virus. However, whereas sample transfer provides higher sensitivity for detection of TBEV from solid organs, the plaque assay is less costly and considering animals welfare more convenient. Thus, the newly devised method may allow the resolution of unanswered questions, while both the traditional infectivity assays retain their benefits in certain situations.

Detection of tick-borne encephalitis virus RNA in ticks ( Ixodes ricinus) by the polymerase chain reaction

A polymerase chain reaction for the detection of tick-borne encephalitis virus (TBEV) RNA in ticks was developed. Two pairs of primers for nested PCR were selected from the 5'-NCR and the 5'-terminus of the C protein coding region, which are highly conserved among the TBEV isolates sequenced so far. The sensitivity of the nested PCR was tested by dilution experiments of a TBEV positive brain suspension. The specificity of the PCR products was confirmed by Southern blotting. In a pilot study, 60 homogenates of 7200 ticks ( I. ricinus) were examined by PCR. Two homogenates were found positive. The PCR for TBEV RNA appears to be a valuable method to define endemic areas of TBE.